Use of semaphorin-4D binding molecules for treating neurodegenerative disorders

ABSTRACT

Provided herein are methods for alleviating symptoms in a subject having a neurodegenerative disorder, comprising administering to the subject an effective amount of an isolated binding molecule which specifically binds to semaphorin-4D (SEMA4D) or to its Plexin-B1 or Plexin-B2 receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of and claims the benefit of bothU.S. Non-Provisional patent application Ser. No. 15/420,662, filed onJan. 31, 2017 and U.S. Non-Provisional patent application Ser. No.14/519,965, filed on Oct. 21, 2014, now U.S. Pat. No. 9,598,495, issuedMar. 21, 2017, and also claims benefit to U.S. Provisional applicationNo. 62/012,805, filed on Jun. 16, 2014, U.S. Provisional Appl. No.61/979,384, filed on Apr. 14, 2014, and U.S. Provisional Appl. No.61/893,814, filed on Oct. 21, 2013, the contents of which are eachhereby incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name 58008-163820seq-1stg_Div_ST25.txt; Size: 32,500 bytes;and Date of Creation: Mar. 1, 2017) filed with the application isincorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein(e.g., SEQ ID NO: 1 (human); SEQ ID NO: 2 (murine)) that belongs to thesemaphorin gene family. SEMA4D is expressed on the cell surface as ahomodimer, but upon cell activation SEMA4D can be released from the cellsurface via proteolytic cleavage to generate sSEMA4D, a soluble form ofthe protein, which is also biologically active. See Suzuki et al.,Nature Rev. Immunol. 3:159-167 (2003); Kikutani et al., Nature Immunol.9:17-23 (2008).

SEMA4D is expressed at high levels in lymphoid organs, including thespleen, thymus, and lymph nodes, and in non-lymphoid organs, such as thebrain, heart, and kidney. In lymphoid organs, SEMA4D is abundantlyexpressed on resting T cells but only weakly expressed on resting Bcells and antigen-presenting cells (APCs), such as dendritic cells(DCs). Its expression, however, is upregulated in these cells followingactivation by various immunological stimuli. The release of solubleSEMA4D from immune cells is also increased by cell activation.

SEMA4D has been implicated in the development of neurodegenerativedisorders, autoimmune diseases, demyelinating diseases, and certaincancers. However, the effect of blocking SEMA4D signaling on theorganization and function of the central nervous system (CNS) includingbrain and spinal cord and on behaviors controlled by the CNS remains tobe elucidated. This is important because changes in the CNS have aprofound influence on a subject's behavior and quality of life. Inparticular, such changes can impact a subject's neuropsychiatricbehavior, cognitive behavior, and motor skills. There remains,therefore, a need for treatments for neurodegenerative disorders thatalleviate the symptoms associated with the disorder.

BRIEF SUMMARY OF THE DISCLOSURE

Methods for using semaphorin 4D binding molecules to alleviate symptomsin a subject having neurodegenerative disorders are disclosed herein.According to aspects of the disclosure illustrated herein, there isprovided a method for improving symptoms in a subject with aneurodegenerative disorder including administering to the subject aneffective amount of an isolated binding molecule which specificallybinds to semaphorin 4D (SEMA4D) and inhibits, suppresses, prevents,reverses or slows the effect of SEMA4D.

According to aspects illustrated herein, there is provided a method oftreating a subject with a neurodegenerative disorder includingadministering to the subject an effective amount of an isolated bindingmolecule which specifically binds to semaphorin 4D (SEMA4D), wherein thebinding to SEMA4D acts to improve symptoms associated with the disorder.

Methods of alleviating symptoms in a subject having a neurodegenerativedisorder are provided, comprising administering to that subject aneffective amount of an isolated binding molecule which specificallybinds to semaphorin-4D (SEMA4D). In certain embodiments of the methods,the binding molecule inhibits SEMA4D interaction with its receptor or aportion of its receptor. In certain embodiments of the methods, thereceptor is selected from the group consisting of Plexin-B1 andPlexin-B2. In certain embodiments of the methods, the binding moleculeinhibits SEMA4D-mediated Plexin-B1 signal transduction. In certainembodiments of the methods, the isolated binding molecule specificallybinds to the same SEMA4D epitope as a reference monoclonal antibodyselected from the group consisting of VX15/2503 or 67. In certainembodiments of the methods, the isolated binding molecule competitivelyinhibits a reference monoclonal antibody selected from the groupconsisting of VX15/2503 or 67 from specifically binding to SEMA4D. Incertain embodiments of the methods, the isolated binding moleculecomprises an antibody or antigen-binding fragment thereof. In certainembodiments of the methods, the antibody or antigen-binding fragmentthereof is monoclonal antibody VX15/2503 or 67. In certain embodimentsof the methods, the antibody or antigen-binding fragment thereofcomprises a variable heavy chain (VH) comprising VHCDRs 1-3 comprisingSEQ ID NOs 6, 7, and 8, respectively, and a variable light chain (VL)comprising VLCDRs 1-3 comprising SEQ ID NOs 14, 15, and 16,respectively. In certain embodiments of the methods, the VH and VLcomprise, respectively, SEQ ID NO: 9 and SEQ ID NO: 17 or SEQ ID NO: 10and SEQ ID NO: 18. In certain embodiments of any of the aforementionedmethods, the neurodegenerative disorder is selected from a groupconsisting of Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Down syndrome, ataxia, amyotrophic lateral sclerosis (ALS),frontotemporal dementia (FTD), HIV-related cognitive impairment, CNSLupus, mild cognitive impairment, or a combination thereof. In certainembodiments of any of the aforementioned methods, the neurodegenerativedisorder is Alzheimer's disease or Huntington's disease. In certainembodiments of any one of the aforementioned methods, the symptoms areselected from a group consisting of neuropsychiatric symptoms, cognitivesymptoms, motor dysfunction, and any combination thereof. In certainembodiments of any of the aforementioned methods, the neuropsychiatricsymptoms are selected from a group consisting of reducing anxiety-likebehavior, improving spatial memory, increasing locomotion, and anycombination thereof.

Methods of alleviating symptoms in a subject having a neurodegenerativedisorder are provided, comprising administering to that subject aneffective amount of an isolated binding molecule which specificallybinds to SEMA4D, wherein the binding molecule competitively inhibits areference monoclonal antibody selected from the group consisting ofVX15/2503 or 67 from specifically binding to SEMA4D. In certainembodiments of the methods, the binding molecule inhibits SEMA4Dinteraction with its receptor or a portion of its receptor. In certainembodiments of the methods, the receptor is selected from the groupconsisting of Plexin-B1 and Plexin-B2. In certain embodiments of themethods, the binding molecule inhibits SEMA4D-mediated Plexin-B1 signaltransduction. In certain embodiments of the methods, the isolatedbinding molecule comprises an antibody or antigen-binding fragmentthereof. In certain embodiments of the methods, the antibody orantigen-binding fragment thereof is monoclonal antibody VX15/2503 or 67.In certain embodiments of the methods, the antibody or antigen-bindingfragment thereof comprises a variable heavy chain (VH) comprising VHCDRs1-3 comprising SEQ ID NOs 6, 7, and 8, respectively, and a variablelight chain (VL) comprising VLCDRs 1-3 comprising SEQ ID NOs 14, 15, and16, respectively. In certain embodiments of the methods, the VH and VLcomprise, respectively, SEQ ID NO: 9 and SEQ ID NO: 17 or SEQ ID NO: 10and SEQ ID NO: 18. In certain embodiments of any of the aforementionedmethods, the neurodegenerative disorder is selected from a groupconsisting of Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Down syndrome, ataxia, amyotrophic lateral sclerosis (ALS),frontotemporal dementia (FTD), HIV-related cognitive impairment, CNSLupus, mild cognitive impairment, or a combination thereof. In certainembodiments of any of the aforementioned methods, the neurodegenerativedisorder is Alzheimer's disease or Huntington's disease. In certainembodiments of any one of the aforementioned methods, the symptoms areselected from a group consisting of neuropsychiatric symptoms, cognitivesymptoms, motor dysfunction, and any combination thereof. In certainembodiments of any of the aforementioned methods, the neuropsychiatricsymptoms are selected from a group consisting of reducing anxiety-likebehavior, improving spatial memory, increasing locomotion, and anycombination thereof

Additional methods of treating a subject having a neurodegenerative orneuroinflammatory disorder, or of effecting a desirable outcome in asubject having a neurodegenerative or neuroinflammatory disorder, areprovided herein. Methods of treating a subject having aneurodegenerative disorder are provided, comprising administering to thesubject an effective amount of an isolated binding molecule whichspecifically binds to semaphorin-4D (SEMA4D), wherein the binding toSEMA4D acts to alleviate symptoms associated with the disorder. Methodsof promoting myelination in a subject having a neurodegenerativedisorder are provided, comprising administering to that subject aneffective amount of an isolated binding molecule which specificallybinds to SEMA4D, wherein the binding molecule modulatesastrocyte-mediated activity of oligodendrocyte-myelin function. Methodsof preventing neural cell death in a subject having a neurodegenerativedisorder are provided, comprising administering to that subject aneffective amount of an isolated binding molecule which specificallybinds to SEMA4D, wherein the binding molecule modulatesastrocyte-mediated synaptic activity. Methods of preventing injury tothe blood-brain barrier in a subject having a neuroinflammatory orneurodegenerative disorder are provided, comprising administering tothat subject an effective amount of an isolated binding molecule whichspecifically binds to SEMA4D, wherein the binding molecule modulatesastrocyte-mediated maintenance of the integrity of the blood-brainbarrier. Methods of preventing astrocyte activation in a subject having,determined to have, or suspected of having a neuroinflammatory orneurodegenerative disorder are provided, comprising administering tothat subject an effective amount of an isolated binding molecule whichspecifically binds to SEMA4D, wherein the binding molecule modulatesastrocyte-mediated maintenance of the integrity of the blood-brainbarrier. Methods of maintaining or restoring astrocyte-mediated trophicsupport of oligodendrocyte precursor cells (OPCs) in a subject having,determined to have, or suspected of having a neuroinflammatory orneurodegenerative disorder are provided, comprising administering tothat subject an effective amount of an isolated binding molecule whichspecifically binds to SEMA4D, wherein the binding molecule preventsretraction of astrocyte processes and chemotactic movement of OPCstoward regions of damage. Methods of protecting inhibitory neurons fromdegeneration in early Alzheimer's disease are provided, comprisingadministering to a subject having, determined to have, or suspected ofhaving early Alzheimer's disease an effective amount of an isolatedbinding molecule which specifically binds to SEMA4D, wherein the bindingmolecule restores the number of somatostatin positive neurons,NYP-positive neurons, or both in the subject. In certain embodiments ofthe aforementioned methods, the binding molecule inhibits SEMA4Dinteraction with its receptor or a portion of its receptor. In certainembodiments of the aforementioned methods, the receptor is selected fromthe group consisting of Plexin-B1 and Plexin-B2. In certain embodimentsof the aforementioned methods, the binding molecule inhibitsSEMA4D-mediated Plexin-B1 signal transduction. In certain embodiments ofthe aforementioned methods, the isolated binding molecule specificallybinds to the same SEMA4D epitope as a reference monoclonal antibodyselected from the group consisting of VX15/2503 or 67. In certainembodiments of the aforementioned methods, the isolated binding moleculecompetitively inhibits a reference monoclonal antibody selected from thegroup consisting of VX15/2503 or 67 from specifically binding to SEMA4D.In certain embodiments of the aforementioned methods, the isolatedbinding molecule comprises an antibody or antigen-binding fragmentthereof. In certain embodiments of the aforementioned methods, theantibody or antigen-binding fragment thereof is monoclonal antibodyVX15/2503 or 67. In certain embodiments of the aforementioned methods,the antibody or antigen-binding fragment thereof comprises a variableheavy chain (VH) comprising VHCDRs 1-3 comprising SEQ ID NOs 6, 7, and8, respectively, and a variable light chain (VL) comprising VLCDRs 1-3comprising SEQ ID NOs 14, 15, and 16, respectively. In certainembodiments of the aforementioned methods, the VH and VL comprise,respectively, SEQ ID NO: 9 and SEQ ID NO: 17 or SEQ ID NO: 10 and SEQ IDNO: 18. In certain embodiments of the aforementioned methods, theneurodegenerative disorder is selected from a group consisting ofAlzheimer's disease, Parkinson's disease, Huntington's disease, Downsyndrome, ataxia, amyotrophic lateral sclerosis (ALS), frontotemporaldementia (FTD), HIV-related cognitive impairment, CNS Lupus, mildcognitive impairment, or a combination thereof. In certain embodimentsof the aforementioned methods, the neurodegenerative disorder isAlzheimer's disease or Huntington's disease. In certain embodiments ofthe aforementioned methods, symptoms of the subject that are alleviatedby the methods are selected from a group consisting of neuropsychiatricsymptoms, cognitive symptoms, motor dysfunction, and any combinationthereof. In certain embodiments of the aforementioned methods, theneuropsychiatric symptoms of the subject that are alleviated by themethods are selected from a group consisting of reducing anxiety-likebehavior, improving spatial memory, increasing locomotion, and anycombination thereof. In certain embodiments of the aforementionedmethods, the subject is determined to have the neurodegenerativedisorder by processing a sample or image from the subject.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Schematic of experimental protocol described in the Examples.

FIG. 2A: In vivo CVN model measuring anxiety-like behavior in CVN micetreated with anti-SEMA4D antibody (“MAb 67”) or control isotype (“MAb2B8”), total locomotion.

FIG. 2B: In vivo CVN model measuring anxiety-like behavior in CVN micetreated with anti-SEMA4D antibody (“MAb 67”) or control isotype (“MAb2B8”), locomotion in the center of the open field.

FIG. 3A: a photograph of a redial-arm water maze.

FIG. 3B: In vivo CVN model measuring spatial memory in a radial-armwater maze, CVN mice treated with anti-SEMA4D antibody (“MAb 67”) orcontrol isotype.

FIG. 4A: In vivo CVN model measuring the density of GABAergic synapsesand concentration of vesicular GABA transporter (VGAT) in CVN micetreated with anti-SEMA4D antibody (“MAb 67”) or control isotype, graphshowing VGAT positive vesicles.

FIG. 4B: In vivo CVN model measuring the density of GABAergic synapsesand concentration of vesicular GABA transporter (VGAT) in CVN micetreated with anti-SEMA4D antibody (“MAb 67”) or control isotype, graphshowing VGAT staining intensity level per vesicle.

FIG. 5A: In vivo YAC128 model measuring anxiety-like behavior in micetreated with anti-SEMA4D antibody (“MAb 67”) and control isotype, graphshowing entries into field.

FIG. 5B: In vivo YAC128 model measuring anxiety-like behavior in micetreated with anti-SEMA4D antibody (“MAb 67”) and control isotype, graphshowing time spent in the center of the field.

FIG. 6A: In vivo YAC128 model measuring spatial memory in mice treatedwith anti-SEMA4D antibody (“MAb 67”) and control isotype, Trial 1.

FIG. 6B: In vivo YAC128 model measuring spatial memory in mice treatedwith anti-SEMA4D antibody (“MAb 67”) and control isotype, Trial 2.

FIG. 7A: In vivo YAC128 model measuring cortical volume in mice treatedwith anti-SEMA4D antibody (“MAb 67”) or control isotype.

FIG. 7B: In vivo YAC128 model measuring corpus callosum volume in micetreated with anti-SEMA4D antibody (“MAb 67”) or control isotype.

FIG. 8: In vivo YAC128 model measuring testicular degeneration in micetreated with anti-SEMA4D antibody (“MAb 67”) or control isotype.

FIG. 9A-P: Immunohistochemical analysis of cell types expressing SEMA4D,plexin-B1, and CD72 in normal rat spinal cord.

FIG. 9B: Nkx2.2 is an oligodendrocyte precursor cell marker.

FIG. 9G: glial fibrillary acid protein (GFAP) is an astrocytic cellmarker.

FIG. 9N: Iba1 (panel N) is a microglial cell marker. FIGS. 9A, 9E, 9I,and 9M show merged images, and FIGS. 9D, 9H, 9L, and 9P, show the samesections stained with DAPI to visualize cellular nuclei.

FIG. 10: DAB immunohistochemical analysis of amyloid pathology and glialactivation in normal (top three panels) and CVN (bottom three panels)mice. Subiculum sections were stained for amyloid-beta 1-42 (leftpanels), the microglial cell marker Iba1 (middle panels) and theastrocytic cell marker GFAP.

FIG. 11A: Characterization and expression patterns of plexin-B1 andplexin-B2 receptors in the CVN Alzheimer's disease mouse model, showingimmunohistochemical analysis of plexin-B1 expression in normal (toppanels) and CVN (bottom panels) mice. Brain sections were stained forplexin-B1, and GFAP, as well as DAPI to visualize cellular nuclei.

FIG. 11B: Characterization and expression patterns of plexin-B1 andplexin-B2 receptors in the CVN Alzheimer's disease mouse model, showingexpression levels of plexin-B1 (left graph) and plexin-B2 (right graph)following inhibition of SEMA4D signaling.

FIG. 12: Immunohistochemical analysis of plexin-B2 expression in normal(top panels) and YAC128 (bottom panels) mice. Brain sections werestained for plexin-B2, and GFAP, as well as DAPI to visualize cellularnuclei.

FIG. 13: Schematic representation of the roles SEMA4D signaling can playin the regulation of astrocyte function in health and disease. LeftPanel: Plexin+ (shaded region of astrocyte exterior surface) astrocyticprocesses interdigitate between SEMA4D+ NIKX2.2+ oligodendrocyteprecursor cells (OPCs) and provide trophic support (SEMA4D+ shown asshaded region of OPC exterior surface). In CNS disease, activatedastrocytes upregulate Plexin expression and retract processes via SEMA4Dsignaling. Locally, this results in diminished trophic support andincreased chemotaxis-driven OPC movement toward regions of damage, whilelack of astrocytic support at lesion site impedes remyelination. CenterPanel: In CNS disease, astrocytic activation leads to upregulation ofPlexin (shaded region of astrocyte exterior surface) expression,increased SEMA4D signaling and process retraction, which results in aloss of neuronal axon guidance, decreased trophic support, and/ordysregulated glutamate uptake/release. Ultimately, depending uponseverity of disease stimulus, synapse loss and subsequent excitotoxicneuron death can occur. Right Panel: CNS disease-induced astrocyteactivation increases SEMA4D signaling through Plexin (shaded region ofastrocyte exterior surface), which leads to a retraction of astrocyticfoot processes as evidenced by redistribution of aquaporin-4. Thisresults in dysregulation and permeability of the BBB, therebyfacilitating endothelial inflammation and subsequent leukocyte entryinto the CNS.

FIG. 14: Immunohistochemical analysis showing SEMA4D-expressing OPCsoriented in close association with GFAP+ astrocytic processes in normalrats. Brain sections were stained for SEMA4D (OPCs), and GFAP(astrocytes), as well as DAPI to visualize cellular nuclei.

FIG. 15A: In vivo CVN model measuring somatostatin-positive signalingwithin the subiculum or dentate gyrus in CVN mice treated withanti-SEMA4D antibody (“MAb 67”) or control isotype. Error bars indicatestandard error. “*”=p<0.05 and “***”=p<0.005 by 1-way ANOVA withBonferroni's Multiple Comparison Test.

FIG. 15B: In vivo CVN model measuring neuropeptide-Y (NPY)-positivesignaling within the subiculum or dentate gyrus, respectively, in CVNmice treated with anti-SEMA4D antibody (“MAb 67”) or control isotype.Error bars indicate standard error. “*”=p<0.05 and “***”=p<0.005 by1-way ANOVA with Bonferroni's Multiple Comparison Test.

FIG. 15C: In vivo CVN model measuring NPY receptor 1 (NPY1R) positivesignaling within the subiculum or dentate gyrus in CVN mice treated withanti-SEMA4D antibody (“MAb 67”) or control isotype. Error bars indicatestandard error. “*”=p<0.05 and “***”=p<0.005 by 1-way ANOVA withBonferroni's Multiple Comparison Test.

FIG. 15D: In vivo CVN model measuring NPY receptor 2 (NPY2R) (panel D)positive signaling within the subiculum or dentate gyrus in CVN micetreated with anti-SEMA4D antibody (“MAb 67”) or control isotype. Errorbars indicate standard error. “*”=p<0.05 and “***”=p<0.005 by 1-wayANOVA with Bonferroni's Multiple Comparison Test.

FIG. 16: Immunohistochemical analysis of aquaporin-4 expression patternsin normal and CVN mice.

FIG. 17: In vitro DIV-BBB model measuring integrity of the blood-brainbarrier upon addition of anti-SEMA4D monoclonal antibody VX15/2503.

FIG. 18A: Immunocytochemical analysis showing astrocyte activation inrat astrocytes, showing immunocytochemical analysis of astrocyteactivation as reflected in the relative increase in GFAP positive areain cultured rat astrocytes treated with SEMA4D in isolation or followingpretreatment with thioacetamide (TAA). “*”=P<0.05 by one-way ANOVA withBonferroni's Multiple Comparison Test.

FIG. 18B: Immunocytochemical analysis showing astrocyte activation inrat astrocytes, showing immunocytochemical analysis of astrocyteactivation as reflected in the ratio of F-actin to G-actin in culturedrat astrocytes treated with SEMA4D in isolation or in combination withprostaglandin D2. Error bars represent standard deviation. “*”=P<0.05 byone-way ANOVA with Bonferroni's Multiple Comparison Test.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an anti-SEMA4D antibody” is understood torepresent one or more anti-SEMA4D antibodies. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

As used herein, the term “non-naturally occurring” substance,composition, entity, and/or any combination of substances, compositions,or entities, or any grammatical variants thereof, is a conditional termthat explicitly excludes, but only excludes, those forms of thesubstance, composition, entity, and/or any combination of substances,compositions, or entities that are well-understood by persons ofordinary skill in the art as being “naturally-occurring,” or that are,or might be at any time, determined or interpreted by a judge or anadministrative or judicial body to be, “naturally-occurring.”

As used herein, the term “neurodegenerative disorder” or“neurodegenerative disease” refers to a central nervous system (CNS)disorder that is characterized by the death of neurons in one or moreregions of the nervous system and the subsequent functional impairmentof the affected parties. Examples of neurodegenerative disordersinclude, without limitation, Alzheimer's disease, Parkinson's disease,Huntington's disease, Down syndrome, ataxia, amyotrophic lateralsclerosis (ALS), frontotemporal dementia (FTD), HIV-related cognitiveimpairment (HAND, HIV-Associated Neurocognitive Disorder), CNS Lupus andmild cognitive impairment. Neurodegenerative diseases have an enormousimpact on the lives of affected individuals and their families as wellas society as a whole.

As used herein, the term “Alzheimer's disease” refers to a progressivedisease initially manifesting itself with partial amnesia, and laterrestlessness, disorientation, aphasia, agnosia or apraxia (cognitivedecline), dementia and sometimes euphoria or depressions. The diseasetypically starts at 40 to 90 years of age and predominantly affectsfemales. As to its prevalence, estimations are about 13% of thepopulation above 65 years age.

As used herein, the term “Huntington's Disease” refers to aneurodegenerative disease, which is due to expansion of a poly-glutaminetract at the N-terminus of the protein huntingtin (expressed by the HTTgene) where the expansion can be more than 35-40 repetitions of theamino acid glutamine in the mutated protein (mHTT). The disease presentswith progressive neuronal death in different brain areas, includingtoxicity in medium-sized spiny neurons of the striatum that determinesthe appearance of the classic motor incoordination and movements such as“Chorea”. The mechanism of action of mHTT has been described as bothgain and loss of function compared with the wild-type protein andinvolves the acquisition or loss of competence to interact with variousproteins in different cellular compartments.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide, polynucleotide, small organic molecule, or otherdrug effective to “treat” a disease or disorder in a subject or mammal.In the case of a neurodegenerative disorder, the therapeuticallyeffective amount of the drug can alleviate symptoms of the disorder;decrease, reduce, retard or stop the incidence of symptoms; decrease,reduce, retard the severity of symptoms; inhibit, e.g., suppress,retard, prevent, stop, or reverse the manifestation of symptoms; relieveto some extent one or more of the symptoms associated with the disorder;reduce morbidity and mortality; improve quality of life; or acombination of such effects.

The term “symptoms” as referred to herein refer to, e.g., 1)neuropsychiatric symptoms, 2) cognitive symptoms, and 3) motordysfunction. Examples of neuropsychiatric symptoms include, forinstance, anxiety-like behavior. Examples of cognitive symptoms include,for instance, learning and memory deficits. Examples of motordysfunction include, for instance, locomotion.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” or “improving” or “to improve” refer to both 1)therapeutic measures that cure, slow down, lessen symptoms of, reverse,and/or halt progression of a diagnosed pathologic condition or disorderand 2) prophylactic or preventative measures that prevent and/or slowthe development of a targeted pathologic condition or disorder. Thusthose in need of treatment include those already with the disorder;those prone to have the disorder; and those in whom the disorder is tobe prevented. Beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilization (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows,bears, and so on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-SEMA4D antibody” and “an animal in need oftreatment” includes subjects, such as mammalian subjects, that wouldbenefit from administration of an anti-SEMA4D antibody or other SEMA4Dbinding molecule used, e.g., for detection of a SEMA4D polypeptide(e.g., for a diagnostic procedure) and/or from treatment, i.e.,palliation or prevention of a disease, with an anti-SEMA4D antibody orother SEMA4D binding molecule.

A “binding molecule” or “antigen binding molecule” of the presentdisclosure refers in its broadest sense to a molecule that specificallybinds an antigenic determinant. In one embodiment, the binding moleculespecifically binds to SEMA4D, e.g., to a transmembrane SEMA4Dpolypeptide of about 150 kDa or a soluble SEMA4D polypeptide of about120 kDa (commonly referred to as sSEMA4D). In another embodiment, abinding molecule of the disclosure is an antibody or an antigen bindingfragment thereof. In another embodiment, a binding molecule of thedisclosure comprises at least one heavy or light chain CDR of anantibody molecule. In another embodiment, a binding molecule of thedisclosure comprises at least two CDRs from one or more antibodymolecules. In another embodiment, a binding molecule of the disclosurecomprises at least three CDRs from one or more antibody molecules. Inanother embodiment, a binding molecule of the disclosure comprises atleast four CDRs from one or more antibody molecules. In anotherembodiment, a binding molecule of the disclosure comprises at least fiveCDRs from one or more antibody molecules. In another embodiment, abinding molecule of the disclosure comprises at least six CDRs from oneor more antibody molecules.

The present disclosure is directed to a method of alleviating symptomsin a subject having a neurodegenerative disorder, comprisingadministering to the subject an anti-SEMA4D binding molecule, e.g., anantibody, or antigen-binding fragment, variant, or derivative thereof.Unless specifically referring to full-sized antibodies such as naturallyoccurring antibodies, the term “anti-SEMA4D antibody” encompassesfull-sized antibodies as well as antigen-binding fragments, variants,analogs, or derivatives of such antibodies, e.g., naturally occurringantibody or immunoglobulin molecules or engineered antibody molecules orfragments that bind antigen in a manner similar to antibody molecules.

As used herein, “human” or “fully human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example, in U.S.Pat. No. 5,939,598 by Kucherlapati et al. “Human” or “fully human”antibodies also include antibodies comprising at least the variabledomain of a heavy chain, or at least the variable domains of a heavychain and a light chain, where the variable domain(s) have the aminoacid sequence of human immunoglobulin variable domain(s).

“Human” or “fully human” antibodies also include “human” or “fullyhuman” antibodies, as described above, that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind toa SEMA4D polypeptide or fragment or variant thereof. Standard techniquesknown to those of skill in the art can be used to introduce mutations inthe nucleotide sequence encoding a human anti-SEMA4D antibody,including, but not limited to, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions. Insome embodiments, the variants (including derivatives) encode less than50 amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference VH region, VHCDR1, VHCDR2, VHCDR3, VL region,VLCDR1, VLCDR2, or VLCDR3.

In certain embodiments, the amino acid substitutions are conservativeamino acid substitutions, discussed further below. Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind a SEMA4D polypeptide, e.g., human,murine, or both human and murine SEMA4D). Such variants (or derivativesthereof) of “human” or “fully human” antibodies can also be referred toas human or fully human antibodies that are “optimized” or “optimizedfor antigen binding” and include antibodies that have improved affinityto antigen.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

As used herein, the term “immunoglobulin” comprises various broadclasses of polypeptides that can be distinguished biochemically. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with somesubclasses among them (e.g., γ1-γ4 γ4. γ4). It is the nature of thischain that determines the “class” of the antibody as IgG, IgM, IgA IgG,or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g.,IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. are well characterized and areknown to confer functional specialization. Modified versions of each ofthese classes and isotypes are readily discernable to the skilledartisan in view of the instant disclosure and, accordingly, are withinthe scope of the instant disclosure. All immunoglobulin classes areclearly within the scope of the present disclosure, the followingdiscussion will generally be directed to the IgG class of immunoglobulinmolecules. With regard to IgG, a standard immunoglobulin moleculecomprises two identical light chain polypeptides of molecular weightapproximately 23,000 Daltons, and two identical heavy chain polypeptidesof molecular weight 53,000-70,000. The four chains are typically joinedby disulfide bonds in a “Y” configuration wherein the light chainsbracket the heavy chains starting at the mouth of the “Y” and continuingthrough the variable region.

Light chains are classified as either kappa or lambda (κ, λ) Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL or VK) and heavy (VH) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (typically CH1, CH2or CH3) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the antibody. The N-terminal portion is a variableregion and at the C-terminal portion is a constant region; the CH3 andCL domains typically comprise the carboxy-terminus of the heavy andlight chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to form the variable region that defines a three dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. In some instances, e.g., certain immunoglobulinmolecules derived from camelid species or engineered based on camelidimmunoglobulins, a complete immunoglobulin molecule can consist of heavychains only, with no light chains. See, e.g., Hamers-Casterman et al.,Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art, since they have been preciselydefined (see below).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest” and byChothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residuesthat encompass the CDRs as defined by each of the above cited referencesare set forth below in Table 1 as a comparison. The exact residuenumbers that encompass a particular CDR will vary depending on thesequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.” Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an anti-SEMA4D antibody orantigen-binding fragment, variant, or derivative thereof of the presentdisclosure are according to the Kabat numbering system.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the disclosure include, but are not limited to, polyclonal,monoclonal, multispecific and bispecific in which at least one arm isspecific for SEMA4D, human, humanized, primatized, or chimericantibodies, single-chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv),disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto anti-SEMA4D antibodies disclosed herein). ScFv molecules are known inthe art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the disclosure can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulinmolecule.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. In certainembodiments, a polypeptide comprising a heavy chain portion comprises atleast one of: a VH domain, a CH1 domain, a hinge (e.g., upper, middle,and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or avariant or fragment thereof. For example, a binding polypeptide for usein the disclosure can comprise a polypeptide chain comprising a CH1domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH2 domain; a polypeptide chain comprising aCH1 domain and a CH3 domain; a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, and a CH3 domain, or apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, a CH2 domain, and a CH3 domain. In another embodiment, apolypeptide of the disclosure comprises a polypeptide chain comprising aCH3 domain. Further, a binding polypeptide for use in the disclosure canlack at least a portion of a CH2 domain (e.g., all or part of a CH2domain). As set forth above, it will be understood by one of ordinaryskill in the art that these domains (e.g., the heavy chain portions) canbe modified such that they vary in amino acid sequence from thenaturally occurring immunoglobulin molecule.

In certain anti-SEMA4D antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein, the heavy chainportions of one polypeptide chain of a multimer are identical to thoseon a second polypeptide chain of the multimer. Alternatively, heavychain portion-containing monomers of the disclosure are not identical.For example, each monomer can comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding molecule for use in the methodsdisclosed herein can be derived from different immunoglobulin molecules.For example, a heavy chain portion of a polypeptide can comprise aC_(H1) domain derived from an IgG1 molecule and a hinge region derivedfrom an IgG3 molecule. In another example, a heavy chain portion cancomprise a hinge region derived, in part, from an IgG1 molecule and, inpart, from an IgG3 molecule. In another example, a heavy chain portioncan comprise a chimeric hinge derived, in part, from an IgG1 moleculeand, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain, e.g., a kappa orlambda light chain. In some aspects, the light chain portion comprisesat least one of a VL or CL domain.

Anti-SEMA4D antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein can be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide disclosed herein (e.g., SEMA4D) that they recognize orspecifically bind. The portion of a target polypeptide that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target polypeptide cancomprise a single epitope, but typically comprises at least twoepitopes, and can include any number of epitopes, depending on the size,conformation, and type of antigen. Furthermore, it should be noted thatan “epitope” on a target polypeptide can be or can includenon-polypeptide elements, e.g., an epitope can include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes can contain, e.g., at least seven, at least nine or between atleast about 15 to about 30 amino acids. Since a CDR can recognize anantigenic peptide or polypeptide in its tertiary form, the amino acidscomprising an epitope need not be contiguous, and in some cases, can beon separate peptide chains. A peptide or polypeptide epitope recognizedby anti-SEMA4D antibodies of the present disclosure can contain asequence of at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 contiguous or non-contiguous amino acids ofSEMA4D.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” can be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” can be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody cancross-react with the related epitope.

By way of non-limiting example, an antibody can be considered to bind afirst epitope preferentially if it binds the first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody can beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody can be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody can be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody can be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody can be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope. An antibody or antigen-binding fragment, variant, orderivative disclosed herein can be said to bind a target polypeptidedisclosed herein (e.g., SEMA4D, e.g., human, murine, or both human andmurine SEMA4D) or a fragment or variant thereof with an off rate(k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹or 10⁻³ sec⁻¹. In certain aspects, an antibody of the disclosure can besaid to bind a target polypeptide disclosed herein (e.g., SEMA4D, e.g.,human, murine, or both human and murine SEMA4D) or a fragment or variantthereof with an off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹,10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹,5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

An antibody or antigen-binding fragment, variant, or derivativedisclosed herein can be said to bind a target polypeptide disclosedherein (e.g., SEMA4D, e.g., human, murine, or both human and murineSEMA4D) or a fragment or variant thereof with an on rate (k(on)) ofgreater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹or 5×10⁴ M⁻¹ sec⁻¹. In some embodiments, an antibody of the disclosurecab be said to bind a target polypeptide disclosed herein (e.g., SEMA4D,e.g., human, murine, or both human and murine SEMA4D) or a fragment orvariant thereof with an on rate (k(on)) greater than or equal to 10⁵ M⁻¹sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition can be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody can be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Anti-SEMA4D antibodies or antigen-binding fragments, variants, orderivatives thereof of the disclosure can also be described or specifiedin terms of their cross-reactivity. As used herein, the term“cross-reactivity” refers to the ability of an antibody, specific forone antigen, to react with a second antigen; a measure of relatednessbetween two different antigenic substances. Thus, an antibody is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, can actually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody can be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody can be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Anti-SEMA4D binding molecules, e.g., antibodies or antigen-bindingfragments, variants or derivatives thereof, of the disclosure can alsobe described or specified in terms of their binding affinity to apolypeptide of the disclosure, e.g., SEMA4D, e.g., human, murine, orboth human and murine SEMA4D. In certain aspects, the binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. In certain embodiments, theanti-SEMA4D binding molecule, e.g., an antibody or antigen bindingfragment thereof, of the disclosure binds human SEMA4D with a Kd ofabout 5×10⁻⁹ to about 6×10⁻⁹. In another embodiment, the anti-SEMA4Dbinding molecule, e.g., an antibody or antigen binding fragment thereof,of the disclosure binds murine SEMA4D with a Kd of about 1×10⁻⁹ to about2×10⁻⁹.

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which can beintact, partial or modified in accordance with the instant disclosure)is obtained from a second species. In certain embodiments the targetbinding region or site will be from a non-human source (e.g., mouse orprimate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class, orfrom an antibody from a different species. An engineered antibody inwhich one or more “donor” CDRs from a non-human antibody of knownspecificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It is not alwaysnecessary to replace all of the CDRs with the complete CDRs from thedonor variable domain to transfer the antigen binding capacity of onevariable domain to another. Rather, one can transfer just those residuesneeded to maintain the activity of the target binding site need betransferred.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody cancomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions” (for example, MAbs 1515/2503 or 67, disclosed in U.S.Patent Appl. Publication No. US 2010/0285036 A1 as MAb 2503,incorporated herein by reference in its entirety). Alternatively, one ormore residues of the framework region(s) of the donor variable domaincan be engineered within the corresponding position of the humanframework region(s) of a variable domain in a heavy or light chain, orboth, of a humanized antibody if necessary to maintain proper binding orto enhance binding to the SEMA4D antigen. A human framework region thathas been engineered in this manner would thus comprise a mixture ofhuman and donor framework residues, and is referred to herein as a“partially human framework region.”

For example, humanization of an anti-SEMA4D antibody can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentor mutant rodent CDRs or CDR sequences for the corresponding sequencesof a human anti-SEMA4D antibody. See also U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated byreference. The resulting humanized anti-SEMA4D antibody would compriseat least one rodent or mutant rodent CDR within the fully humanframework regions of the variable domain of the heavy and/or light chainof the humanized antibody. In some instances, residues within theframework regions of one or more variable domains of the humanizedanti-SEMA4D antibody are replaced by corresponding non-human (forexample, rodent) residues (see, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762; and 6,180,370), in which case the resultinghumanized anti-SEMA4D antibody would comprise partially human frameworkregions within the variable domain of the heavy and/or light chain.

Furthermore, humanized antibodies can comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature 331:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992); herein incorporated by reference.Accordingly, such “humanized” antibodies can include antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some framework residues are substitutedby residues from analogous sites in rodent antibodies. See, for example,U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205.See also U.S. Pat. No. 6,180,370, and International Publication No. WO01/27160, where humanized antibodies and techniques for producinghumanized antibodies having improved affinity for a predeterminedantigen are disclosed.

As used herein, the term “healthcare provider” refers to individuals orinstitutions that directly interact and administer to living subjects,e.g., human patients. Non-limiting examples of healthcare providersinclude doctors, nurses, technicians, therapist, pharmacists,counselors, alternative medicine practitioners, medical facilities,doctor's offices, hospitals, emergency rooms, clinics, urgent carecenters, alternative medicine clinics/facilities, and any other entityproviding general and/or specialized treatment, assessment, maintenance,therapy, medication, and/or advice relating to all, or any portion of, apatient's state of health, including but not limited to general medical,specialized medical, surgical, and/or any other type of treatment,assessment, maintenance, therapy, medication and/or advice.

As used herein, the term “healthcare benefits provider” encompassesindividual parties, organizations, or groups providing, presenting,offering, paying for in whole or in part, or being otherwise associatedwith giving a patient access to one or more healthcare benefits, benefitplans, health insurance, and/or healthcare expense account programs.

As used herein, the term “clinical laboratory” refers to a facility forthe examination or processing of materials or images derived from aliving subject, e.g., a human being. Non-limiting examples of processinginclude biological, biochemical, serological, chemical,immunohematological, hematological, biophysical, cytological,pathological, genetic, image based, or other examination of materialsderived from the human body or of any or all of the human body for thepurpose of providing information, e.g., for the diagnosis, prevention,or treatment of any disease or impairment of, or the assessment of thehealth of living subjects, e.g., human beings. These examinations canalso include procedures to collect or otherwise obtain an image, asample, prepare, determine, measure, or otherwise describe the presenceor absence of various substances in the body of a living subject, e.g.,a human being, or a sample obtained from the body of a living subject,e.g., a human being.

II. Target Polypeptide Description

As used herein, the terms “Semaphorin 4D,” “SEMA4D” and “SEMA4Dpolypeptide” are used interchangeably, as are “SEMA4D” and “Sema4D.” Incertain embodiments, SEMA4D is expressed on the surface of or secretedby a cell. In another embodiment, SEMA4D is membrane bound. In anotherembodiments, SEMA4D is soluble, e.g., sSEMA4D. In other embodiments,SEMA4D can include a full-sized SEMA4D or a fragment thereof, or aSEMA4D variant polypeptide, wherein the fragment of SEMA4D or SEMA4Dvariant polypeptide retains some or all functional properties of thefull-sized SEMA4D.

The full-sized human SEMA4D protein is a homodimeric transmembraneprotein consisting of two polypeptide chains of 150 kDa. SEMA4D belongsto the semaphorin family of cell surface receptors and is also referredto as CD100. Both human and mouse SEMA4D/Sema4D are proteolyticallycleaved from their transmembrane form to generate 120-kDa soluble forms,indicating the existence of two Sema4D isoforms (Kumanogoh et al., J.Cell Science 116(7):3464 (2003)). Semaphorins consist of soluble andmembrane-bound proteins that were originally defined as axonal-guidancefactors during development which play an important role in establishingprecise connections between neurons and their appropriate target.Structurally considered a class IV semaphorin, SEMA4D consists of anamino-terminal signal sequence followed by a characteristic ‘Sema’domain, which contains 17 conserved cysteine residues, an Ig-likedomain, a lysine-rich stretch, a hydrophobic transmembrane region, and acytoplasmic tail.

A polypeptide chain of SEMA4D can include a signal sequence of about 13amino acids and further includes a semaphorin domain of about 512 aminoacids, an immunoglobulin-like (Ig-like) domain of about 65 amino acids,a lysine-rich stretch of 104 amino acids, a hydrophobic transmembraneregion of about 19 amino acids, and a cytoplasmic tail of 110 aminoacids. A consensus site for tyrosine phosphorylation in the cytoplasmictail supports the predicted association of SEMA4D with a tyrosine kinase(Schlossman, et al., Eds. (1995) Leucocyte Typing V (Oxford UniversityPress, Oxford).

SEMA4D is known to have at least three functional receptors, Plexin-B1,Plexin-B2 and CD72. One of the receptors, Plexin-B1, is expressed innon-lymphoid tissues and has been shown to be a high affinity (1 nM)receptor for SEMA4D (Tamagnone et al., Cell 99:71-80 (1999)). SEMA4Dstimulation of Plexin-B1 signaling has been shown to induce growth conecollapse of neurons, and to induce process extension collapse andapoptosis of oligodendrocytes (Giraudon et al., J. Immunol.172:1246-1255 (2004); Giraudon et al., NeuroMolecular Med. 7:207-216(2005)). After binding to SEMA4D, Plexin-B1 signaling mediates theinactivation of R-Ras, leading to a decrease in the integrin mediatedattachment to the extracellular matrix, as well as to activation ofRhoA, leading to reorganization of the cytoskeleton and cell migration.See Kruger et al., Nature Rev. Mol. Cell Biol. 6:789-800 (2005);Pasterkamp, TRENDS in Cell Biology 15:61-64 (2005)). Plexin-B2, on theother hand, has an intermediate affinity for SEMA4D and recent reportsindicate that Plexin-B2 regulates migration of cortical neurons andproliferation and migration of neuroblasts in the adult subventricularzone (Azzarelli et al, Nat Commun 2014 Feb. 27, 5:3405, DOI:10.1038/ncomms4405; and Saha et al., J. Neuroscience, 2012 Nov. 21,32(47): 16892-16905).

In lymphoid tissues CD72 is utilized as a low affinity (300 nM) SEMA4Dreceptor (Kumanogoh et al., Immunity 13:621-631 (2000)). B cells andAPCs express CD72, and anti-CD72 antibodies have many of the sameeffects as sSEMA4D, such as enhancement of CD40-induced B cell responsesand B cell shedding of CD23. CD72 is thought to act as a negativeregulator of B cell responses by recruiting the tyrosine phosphataseSHP-1, which can associate with many inhibitory receptors. Interactionof SEMA4D with CD72 results in the dissociation of SHP-1, and the lossof this negative activation signal. SEMA4D has been reported to promoteT cell stimulation and B cell aggregation and survival in vitro. Theaddition of SEMA4D-expressing cells or sSEMA4D enhances CD40-induced Bcell proliferation and immunoglobulin production in vitro, andaccelerates in vivo antibody responses (Ishida et al., Inter. Immunol.15:1027-1034 (2003); Kumanogoh and H. Kukutani, Trends in Immunol.22:670-676 (2001)). sSEMA4D enhances the CD40 induced maturation ofdendritic cells (DCs), including up-regulation of costimulatorymolecules and increased secretion of IL-12. In addition, sSEMA4D caninhibit immune cell migration, which can be reversed by addition ofblocking anti-SEMA4D antibodies (Elhabazi et al., J. Immunol.166:4341-4347 (2001); Delaire et al., J. Immunol. 166:4348-4354 (2001)).

Sema4D is expressed at high levels in lymphoid organs, including thespleen, thymus, and lymph nodes, and in non-lymphoid organs, such as thebrain, heart, and kidney. In lymphoid organs, Sema4D is abundantlyexpressed on resting T cells but only weakly expressed on resting Bcells and antigen-presenting cells (APCs), such as DCs. Cellularactivation increases the surface expression of SEMA4D as well as thegeneration of soluble SEMA4D (sSEMA4D).

The expression pattern of SEMA4D suggests that it plays an importantphysiological as well as pathological role in the immune system. SEMA4Dhas been shown to promote B cell activation, aggregation and survival;enhance CD40-induced proliferation and antibody production; enhanceantibody response to T cell dependent antigens; increase T cellproliferation; enhance dendritic cell maturation and ability tostimulate T cells; and is directly implicated in demyelination andaxonal degeneration (Shi et al., Immunity 13:633-642 (2000); Kumanogohet al., J Immunol 169:1175-1181 (2002); and Watanabe et al., J Immunol167:4321-4328 (2001)).

SEMA4D knock out (SEMA4D−/−) mice have provided additional evidence thatSEMA4D plays an important role in both humoral and cellular immuneresponses. There are no known major abnormalities of non-lymphoidtissues in SEMA4D−/− mice. DCs from the SEMA4D−/− mice have poorallostimulatory ability and show defects in expression of costimulatorymolecules, which can be rescued by the addition of sSEMA4D. Micedeficient in SEMA4D (SEMA4D−/−) fail to develop experimental autoimmuneencephalomyelitis induced by myelin oligodendrocyte glycoproteinpeptide, because myelin oligodendrocyte glycoprotein-specific T cellsare poorly generated in the absence of SEMA4D (Kumanogoh et al., JImmunol 169:1175-1181 (2002)). A significant amount of soluble SEMA4D isalso detected in the sera of autoimmunity-prone MRL/lpr mice (model ofsystemic autoimmune diseases such as SLE), but not in normal mice.Further, the levels of sSEMA4D correlate with levels of auto-antibodiesand increase with age (Wang et al., Blood 97:3498-3504 (2001)). SolubleSEMA4D has also been shown to accumulate in the cerebral spinal fluidand sera of patients with demyelinating disease, and sSEMA4D inducesapoptosis of human pluripotent neural precursors (Dev cells), and bothinhibit process extension and induce apoptosis of rat oligodendrocytesin vitro (Giraudon et al., J Immunol 172(2):1246-1255 (2004)). Thisapoptosis was blocked by an anti-SEMA4D MAb.

III. Anti-SEMA4D Antibodies

Antibodies that bind SEMA4D have been described in the art. See, forexample, U.S. Pat. No. 8,496,938, US Publ. Nos. 2008/0219971 A1, US2010/0285036 A1, and US 2006/0233793 A1, International PatentApplications WO 93/14125, WO 2008/100995, and WO 2010/129917, and Heroldet al., Int. Immunol. 7(1): 1-8 (1995), each of which is hereinincorporated in its entirety by reference.

The disclosure generally relates to a method of alleviating symptoms ina subject having a neuroinflammatory or neurodegenerative disorder,e.g., a human patient, comprising administration of an antibody whichspecifically binds to SEMA4D, or an antigen-binding fragment, variant,or derivative thereof. In certain embodiments, the antibody blocks theinteraction of SEMA4D with one or more of its receptors, e.g.,Plexin-B1. Anti-SEMA4D antibodies having these properties can be used inthe methods provided herein. Antibodies that can be used include, butare not limited to MAbs VX15/2503, 67, and 76 and antigen-bindingfragments, variants, or derivatives thereof which are fully described inUS 2010/0285036 A1. Additional antibodies which can be used in themethods provided herein include the BD16 and BB18 antibodies describedin US 2006/0233793 A1 as well as antigen-binding fragments, variants, orderivatives thereof; or any of MAb 301, MAb 1893, MAb 657, MAb 1807, MAb1656, MAb 1808, Mab 59, MAb 2191, MAb 2274, MAb 2275, MAb 2276, MAb2277, MAb 2278, MAb 2279, MAb 2280, MAb 2281, MAb 2282, MAb 2283, MAb2284, and MAb 2285, as well as any fragments, variants or derivativesthereof as described in US 2008/0219971 A1. In certain embodiments ananti-SEMA4D antibody for use in the methods provided herein binds human,murine, or both human and murine SEMA4D. Also useful are antibodieswhich bind to the same epitope as any of the aforementioned antibodiesand/or antibodies which competitively inhibit any of the aforementionedantibodies.

In certain embodiments, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein has an amino acid sequence that has at least about 80%, about85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, or about 95% sequence identity to the amino acid sequence fora reference anti-SEMA4D antibody molecule, for example those describedabove. In a further embodiment, the binding molecule shares at leastabout 96%, about 97%, about 98%, about 99%, or 100% sequence identity toa reference antibody.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 9or 10.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to SEQ ID NO: 6, SEQ ID NO: 7, or SEQID NO: 8.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence identical,except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, toSEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of a VH domainthat has an amino acid sequence that is at least about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 9or SEQ ID NO: 10, wherein an anti-SEMA4D antibody comprising the encodedVH domain specifically or preferentially binds to SEMA4D.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO:17 or 18.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to SEQ ID NO: 14, SEQ ID NO: 15, orSEQ ID NO: 16.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence identical,except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, toSEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

In a further embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of a VL domainthat has an amino acid sequence that is at least about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 17or SEQ ID NO: 18, wherein an anti-SEMA4D antibody comprising the encodedVL domain specifically or preferentially binds to SEMA4D.

Also included for use in the methods provided herein are polypeptidesencoding anti-SEMA4D antibodies, or antigen-binding fragments, variants,or derivatives thereof as described herein, polynucleotides encodingsuch polypeptides, vectors comprising such polynucleotides, and hostcells comprising such vectors or polynucleotides, all for producinganti-SEMA4D antibodies, or antigen-binding fragments, variants, orderivatives thereof for use in the methods described herein.

Suitable biologically active variants of the anti-SEMA4D antibodies ofthe disclosure can be used in the methods of the present disclosure.Such variants will retain the desired binding properties of the parentanti-SEMA4D antibody. Methods for making antibody variants are generallyavailable in the art.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Walker and Gaastra, eds. (1983)Techniques in Molecular Biology (MacMillan Publishing Company, NewYork); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel etal., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S.Pat. No. 4,873,192; and the references cited therein; hereinincorporated by reference. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest can be found in the model of Dayhoff et al. (1978) in Atlasof Protein Sequence and Structure (Natl. Biomed. Res. Found, Washington,D.C.), pp. 345-352, herein incorporated by reference in its entirety.The model of Dayhoff et al. uses the Point Accepted Mutation (PAM) aminoacid similarity matrix (PAM 250 matrix) to determine suitableconservative amino acid substitutions. In certain embodiments,conservative substitutions, such as exchanging one amino acid withanother having similar properties can be used. Examples of conservativeamino acid substitutions as taught by the PAM 250 matrix of the Dayhoffet al. model include, but are not limited to, Gly↔Ala, Val↔Ile↔Leu,Asp↔Glu, Lys↔Arg, Asn↔Gln, and Phe↔Trp↔Tyr.

In constructing variants of the anti-SEMA4D binding molecule, e.g., anantibody or antigen-binding fragment thereof, polypeptides of interest,modifications are made such that variants continue to possess thedesired properties, e.g., being capable of specifically binding to aSEMA4D, e.g., human, murine, or both human and murine SEMA4D, e.g.,expressed on the surface of or secreted by a cell and having SEMA4Dblocking activity, as described herein. Obviously, any mutations made inthe DNA encoding the variant polypeptide must not place the sequence outof reading frame and in certain embodiments will not createcomplementary regions that could produce secondary mRNA structure. SeeEP Patent Application Publication No. 75,444.

Methods for measuring anti-SEMA4D binding molecule, e.g., an antibody orantigen-binding fragment, variant, or derivative thereof, bindingspecificity include, but are not limited to, standard competitivebinding assays, assays for monitoring immunoglobulin secretion by Tcells or B cells, T cell proliferation assays, apoptosis assays, ELISAassays, and the like. See, for example, such assays disclosed in WO93/14125; Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., JImmunol 169:1175-1181 (2002); Watanabe et al., J Immunol 167:4321-4328(2001); Wang et al., Blood 97:3498-3504 (2001); and Giraudon et al., JImmunol 172(2):1246-1255 (2004), all of which are herein incorporated byreference.

When discussed herein whether any particular polypeptide, including theconstant regions, CDRs, VH domains, or VL domains disclosed herein, isat least about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or even about 100% identical to anotherpolypeptide, the % identity can be determined using methods and computerprograms/software known in the art such as, but not limited to, theBESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, to find thebest segment of homology between two sequences. When using BESTFIT orany other sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present disclosure, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

For purposes of the present disclosure, percent sequence identity can bedetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math.2:482-489. A variant can, for example, differ from a referenceanti-SEMA4D antibody (e.g., MAb VX15/2503, 67, or 76) by as few as 1 to15 amino acid residues, as few as 1 to 10 amino acid residues, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

Percentage of “sequence identity” can also be determined by comparingtwo optimally aligned sequences over a comparison window. In order tooptimally align sequences for comparison, the portion of apolynucleotide or polypeptide sequence in the comparison window cancomprise additions or deletions termed gaps while the reference sequenceis kept constant. An optimal alignment is that alignment which, evenwith gaps, produces the greatest possible number of “identical”positions between the reference and comparator sequences. Percentage“sequence identity” between two sequences can be determined using theversion of the program “BLAST 2 Sequences” which was available from theNational Center for Biotechnology Information as of Sep. 1, 2004, whichprogram incorporates the programs BLASTN (for nucleotide sequencecomparison) and BLASTP (for polypeptide sequence comparison), whichprograms are based on the algorithm of Karlin and Altschul (Proc. Natl.Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing “BLAST 2Sequences,” parameters that were default parameters as of Sep. 1, 2004,can be used for word size (3), open gap penalty (11), extension gappenalty (1), gap drop-off (50), expect value (10) and any other requiredparameter including but not limited to matrix option.

The constant region of an anti-SEMA4D antibody can be mutated to altereffector function in a number of ways. For example, see U.S. Pat. No.6,737,056B1 and U.S. Patent Application Publication No. 2004/0132101A1,which disclose Fc mutations that optimize antibody binding to Fcreceptors.

In certain anti-SEMA4D antibodies or fragments, variants or derivativesthereof useful in the methods provided herein, the Fc portion can bemutated to decrease effector function using techniques known in the art.For example, the deletion or inactivation (through point mutations orother means) of a constant region domain can reduce Fc receptor bindingof the circulating modified antibody thereby increasing tumorlocalization. In other cases, constant region modifications consistentwith the instant disclosure moderate complement binding and thus reducethe serum half-life. Yet other modifications of the constant region canbe used to modify disulfide linkages or oligosaccharide moieties thatallow for enhanced localization due to increased antigen specificity orantibody flexibility. The resulting physiological profile,bioavailability and other biochemical effects of the modifications, suchas tumor localization, biodistribution and serum half-life, can easilybe measured and quantified using well known immunological techniqueswithout undue experimentation. Anti-SEMA4D antibodies for use in themethods provided herein include derivatives that are modified, e.g., bythe covalent attachment of any type of molecule to the antibody suchthat covalent attachment does not prevent the antibody from specificallybinding to its cognate epitope. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativecan contain one or more non-classical amino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind an anti-SEMA4D polypeptide, to block SEMA4D interactionwith its receptor, or to alleviate symptoms associated with aneurodegenerative disorder in a patient).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations can be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations can be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations can alter an antibody's ability to bind antigen. One of skillin the art would be able to design and test mutant molecules withdesired properties such as no alteration in antigen binding activity oralteration in binding activity (e.g., improvements in antigen bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein can routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of a SEMA4D polypeptide)can be determined using techniques described herein or by routinelymodifying techniques known in the art.

In certain embodiments, the anti-SEMA4D antibodies for use in themethods provided herein comprise at least one optimizedcomplementarity-determining region (CDR). By “optimized CDR” is intendedthat the CDR has been modified and optimized to improve binding affinityand/or anti-SEMA4D activity that is imparted to an anti-SEMA4D antibodycomprising the optimized CDR. “Anti-SEMA4D activity” or “SEMA4D blockingactivity” can include activity which modulates one or more of thefollowing activities associated with SEMA4D: B cell activation,aggregation and survival; CD40-induced proliferation and antibodyproduction; antibody response to T cell dependent antigens; T cell orother immune cell proliferation; dendritic cell maturation;demyelination and axonal degeneration; apoptosis of pluripotent neuralprecursors and/or oligodendrocytes; induction of endothelial cellmigration; inhibition of spontaneous monocyte migration; binding to cellsurface Plexin-B1 or other receptor, or any other activity associatedwith soluble SEMA4D or SEMA4D that is expressed on the surface ofSEMA4D+ cells. Anti-SEMA4D activity can also be attributed to a decreasein incidence or severity of diseases associated with SEMA4D expression,including, but not limited to, certain types of cancers includinglymphomas, autoimmune diseases, inflammatory diseases including centralnervous system (CNS) and peripheral nervous system (PNS) inflammatorydiseases, transplant rejections, and invasive angiogenesis. Examples ofoptimized antibodies based on murine anti-SEMA4D MAbs BD16 and BB18,were described in US Publ. No. 2008/0219971 A1, International PatentApplication WO 93/14125 and Herold et al., Int. Immunol. 7(1): 1-8(1995), each of which are herein incorporated by reference in theirentirety. The modifications can involve replacement of amino acidresidues within the CDR such that an anti-SEMA4D antibody retainsspecificity for the SEMA4D antigen and has improved binding affinityand/or improved anti-SEMA4D activity.

IV. Astrocytes

Astrocytes are specialized glial cells that perform many essentialcomplex functions in the healthy CNS, including regulation of bloodflow, fluid/ion/pH/neurotransmitter homeostasis, synapseformation/function, energy and metabolism, and blood-brain barriermaintenance (Barres B. A. (2008) The mystery and magic of glia: aperspective on their roles in health and disease. Neuron 60:430-440.)Importantly, astrocytes respond to CNS injury through a process referredto as reactive astrogliosis, which serves as a major pathologicalhallmark of neuroinflammatory and neurodegenerative diseases. Increasingevidence points towards the potential of reactive astrogliosis to playeither primary or contributing roles in CNS disorders via loss of normalastrocyte functions or gain of abnormal activities. Given their centralrole in many CNS diseases, there is a significant need to identify andrigorously test new molecular targets that restore normal astrocytefunction to effectively slow or even reverse disease progression. Thereare several potential pathways through which astrocytes can impact CNSdiseases.

Astrocytes and OPC Support.

Demyelination that occurs in neuroinflammatory diseases, such asMultiple Sclerosis, is associated with marked destruction and loss ofcells comprising the oligodendrocyte lineage (Ozawa K, et al. Patternsof oligodendroglia pathology in multiple sclerosis. Brain. 1994;117:1311-1322.). Endogenous remyelination mechanisms fail during therecovery phase in part because of the inability of OPCs to fullydifferentiate into mature myelinating oligodendrocytes (Wolswijk G.Oligodendrocyte survival, loss and birth in lesions of chronic-stagemultiple sclerosis. Brain. 2000; 123:105-115.). Data obtained from otherexperimentally induced demyelination models indicate that newly maturingOPCs, in contrast to surviving mature oligodendrocytes, are required forremyelination during the recovery phase (Levine J M, Reynolds R.Activation and proliferation of endogenous oligodendrocyte precursorcells during ethidium bromide-induced demyelination. Exp Neurol. 1999;160:333-347). Astrocytes have been shown to play a significant role insupporting the function and viability of the oligodendrocyte lineage.For example, Talbott and colleagues showed that in ethidiumbromide-induced demyelinated lesions, astrocytes are required forNkx2.2+/Olig2+ OPCs to fully differentiate into oligodendrocytes andcarry out remyelination (Exp Neurol. 2005 March; 192(1):11-24.Endogenous Nkx2.2+/Olig2+ oligodendrocyte precursor cells fail toremyelinate the demyelinated adult rat spinal cord in the absence ofastrocytes. Talbott J F, Loy D N, Liu Y, Qiu M S, Bunge M B, Rao M S,Whittemore S R). Arai and Lo demonstrated in vitro that astrocytesprovide soluble trophic factor support to OPCs that protect these cellsfrom increased oxidative stress (Arai, K. and Lo, E. H. (2010),Astrocytes protect oligodendrocyte precursor cells via MEK/ERK andPI3K/Akt signaling. J. Neurosci. Res., 88: 758-763. doi:10.1002/jnr.22256). Others have shown that inhibition of astrocyteactivation in the settings of experimental autoimmune encephalomyelitis,experimental optic neuritis, and spinal cord injury leads to improvedremyelination profiles and functional outcome measures (Brambilla R,Persaud T, Hu X, Karmally S, Shestopalov V I, Dvoriantchikova G, IvanovD, Nathanson L, Barnum S R, Bethea J R. 2009. Transgenic inhibition ofastroglial NF-kappa B improves functional outcome in experimentalautoimmune encephalomyelitis by suppressing chronic central nervoussystem inflammation. J Immunol 182:2628-2640; Brambilla R,Dvoriantchikova G, Barakat D, Ivanov D, Bethea J R, Shestopalov V I.2012. Transgenic inhibition of astroglial NF-kappaB protects from opticnerve damage and retinal ganglion cell loss in experimental opticneuritis. J Neuroinflammation 9:213; Brambilla R, Bracchi-Ricard V, Hu WH, Frydel B, Bramwell A, Karmally S, Green E J, Bethea J R. 2005.Inhibition of astroglial nuclear factor kappaB reduces inflammation andimproves functional recovery after spinal cord injury. J Exp Med202:145-156).

Given the role that astrocytes play in facilitation of OPC survival andfunction, the juxtaposition of SEMA4D-expressing OPCs and SEMA4Dreceptor-expressing astrocytes identified here suggests thatdisease-related activation of astrocytes with associated upregulation ofplexin-B receptors and SEMA4D signaling have profound effects on OPCfunction.

Astrocytes and Neuronal Support.

Accumulating evidence indicates that astrocytes play direct roles insynaptic transmission through the regulated release of synapticallyactive molecules including glutamate, purines (ATP and adenosine), GABA,and D-serine (reviewed by Halassa M M, Fellin T, Haydon P G (2007), Thetripartite synapse: roles for gliotransmission in health and disease.Trends Mol Med 13:54-63; Nedergaard M, Ransom B, Goldman S A (2003) Newroles for astrocytes: redefining the functional architecture of thebrain. Trends Neurosci 26:523-530). The release of such gliotransmittersoccurs in response to changes in neuronal synaptic activity, involvesastrocyte excitability as reflected by increases in astrocyte calciumsignaling, and can alter neuronal excitability (Halassa M M, Fellin T,Haydon P G (2007), The tripartite synapse: roles for gliotransmission inhealth and disease. Trends Mol Med 13:54-63; Nedergaard M, Ransom B,Goldman S A (2003) New roles for astrocytes: redefining the functionalarchitecture of the brain. Trends Neurosci 26:523-530). In addition tohaving direct effects on synaptic activity via the release ofgliotransmitters, astrocytes have the potential to exert powerful andlong-term influences on synaptic function through the release of growthfactors and related molecules (Barres B A (2008) The mystery and magicof glia: a perspective on their roles in health and disease. Neuron60:430-440).

Astrocytes and BBB Integrity.

Astrocytes play an essential role in formation of the blood-brainbarrier (BBB) and in regulating transport across the BBB, a homeostaticprocess critical for proper neuronal function. The BBB is a highlycomplex brain endothelial structure of the differentiated neurovascularsystem comprised of pericytes, astrocytes, and endothelial cells. BBBcompromise has been implicated in a number of neurodegenerativediseases, including meningitis, brain edema, epilepsy, Alzheimer'sdisease (AD), Parkinson's disease (PD), stroke, amyotrophic lateralsclerosis (ALS), and Multiple Sclerosis (MS; reviewed by Zlokovic B V.Neurovascular pathways to neurodegeneration in Alzheimer's disease andother disorders. Nat Rev Neurosci. 2011; 12:723-738).

Astrocytes are “polarized” cells in that they extend specializedmembranous processes comprised of unique cellular machinery and membranecomponents that interact with specific cell types. For example,astrocytic processes proximal to cerebral microvessels or pia arecharacterized by a high density of the water channel, aquaporin 4 (Aqp4)(Neely J D, Amiry-Moghaddam M, Ottersen O P, Froehner S C, Agre P, AdamsM E (2001) Syntrophin-dependent expression and localization ofAquaporin-4 water channel protein. Proc Natl Acad Sci USA 98,14108-14113; Amiry-Moghaddam M, Otsuka T, Hurn P D, Traystman R J, HaugF M, Froehner S C, Adams M E, Neely J D, Agre P, Ottersen O P, BhardwajA (2003) An alpha-syntrophin-dependent pool of AQP4 in astroglialend-feet confers bidirectional water flow between blood and brain. ProcNatl Acad Sci USA 100, 2106-2111.). In contrast, astrocytic processesfacing synaptic regions are enriched in glutamate transporters, whilethe density of Aqp4 is comparatively low (Nielsen S, Nagelhus E A,Amiry-Moghaddam M, Bourque C, Agre P, Ottersen O P (1997) Specializedmembrane domains for water transport in glial cells: High-resolutionimmunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci 17,171-180; Chaudhry F A, Lehre K P, van Lookeren Campagne M, Ottersen O P,Danbolt N C, Storm-Mathisen J (1995) Glutamate transporters in glialplasma membranes: Highly differentiated localizations revealed byquantitative ultrastructural immunocytochemistry. Neuron 15, 711-720).Interestingly, astrocytic polarization is disrupted in a brainundergoing neurodegeneration. For example, in the setting of AD, Aqp4staining intensities significantly decrease in regions with significantamyloid plaque burden. In fact, Yang and colleagues showed that theaccumulation of amyloid pathology in tg-ArcSwe AD mice is coupledtemporally and spatially to loss of astrocyte polarization (JAlzheimer's Dis. 2011; 27(4):711-22. doi: 10.3233/JAD-2011-110725; Lossof astrocyte polarization in the tg-ArcSwe mouse model of Alzheimer'sdisease. Yang J L, Lunde L K, Nuntagij P, Oguchi T, Camassa L M, NilssonL N, Lannfelt L, Xu Y, Amiry-Moghaddam M, Ottersen O P, Torp R.).

Role of SEMA4D Signaling in Promoting Astrocyte Activation.

Given the association of SEMA4D receptor expression and the astrocyteactivation marker GFAP, there exists the possibility that SEMA4Dsignaling can potentiate astrocyte activation, thereby providing a“feed-forward” mechanism during disease states. To examine the effectsof SEMA4D on astrocyte activation, primary cultures of rat astrocyteswere generated and treated with SEMA4D in isolation or in combinationwith thioacetamide (TAA) (Example 6 and FIG. 18A below), a well-knownhepatotoxic and hepatocarcinogenic agent that has been shown to induceplexin-B1 expression in vivo (Lim, J. S., Jeong, S. Y., Hwang, J. Y.,Park, H. J., Cho, J. W., & Yoon, S. (2006), or prostaglandin D2 (Example6 and FIG. 18B below), a known activation factor produced by microgliain the CNS, Toxicogenomics Analysis on Thioacetamide-inducedHepatotoxicity in Mice. MOLECULAR & CELLULAR TOXICOLOGY, 2(2),126-133.).

V. Treatment Methods Using Therapeutic Anti-SEMA4D Antibodies

Methods of the disclosure are directed to the use of anti-SEMA4D bindingmolecules, e.g., antibodies, including antigen-binding fragments,variants, and derivatives thereof, to treat a subject having aneurodegenerative disorder. In certain embodiments the endothelial cellsexpress a SEMA4D receptor, in others the neuronal cells express a SEMA4Dreceptor, and in others both endothelial and neuronal cells express aSEMA4D receptor. In certain embodiments the receptor is Plexin-B1.Though the following discussion refers to administration of ananti-SEMA4D antibody, the methods described herein are also applicableto the antigen-binding fragments, variants, and derivatives of theseanti-SEMA4D antibodies or other biologics or small molecules that retainthe desired properties of the anti-SEMA4D antibodies of the disclosure,e.g., capable of specifically binding SEMA4D, e.g., human, mouse, orhuman and mouse SEMA4D, having SEMA4D neutralizing activity, and/orblocking the interaction of SEMA-4D with its receptor, e.g., Plexin-B1.In another embodiment, the methods refers to administration of ananti-SEMA4D antibody, the methods described herein can also refer to theadministration of anti-Plexin-B 1 or anti-Plexin-B2 binding moleculesthat are capable of specifically binding Plexin-B1 and/or Plexin-B2 andblocking the interaction of SEMA-4D with one or both of its Plexinreceptors, e.g., Plexin-B1 and/or Plexin-B2.

In one embodiment, treatment includes the application or administrationof an anti-SEMA4D binding molecule, e.g., an antibody or antigen bindingfragment thereof or other biologic or small molecule that binds andneutralizes SEMA4D as described herein to a patient, where the patienthas, or has the risk of developing a neurodegenerative disorder. Inanother embodiment, treatment is also intended to include theapplication or administration of a pharmaceutical composition comprisingthe anti-SEMA4D binding molecule, e.g., an antibody or antigen bindingfragment thereof to a patient, where the patient has, or has the risk ofdeveloping a neurodegenerative disorder.

The anti-SEMA4D binding molecules, e.g., antibodies or binding fragmentsthereof as described herein are useful for the treatment of variousneurodegenerative disorders. In some embodiments, treatment of aneurodegenerative disorder is intended to induce an improvement in thesymptoms associated with the disorder. In other embodiments, treatmentof a neurodegenerative disorder is intended to reduce, retard or stop anincrease in symptom manifestations. In other embodiments, treatment of aneurodegenerative disorder is intended to inhibit, e.g., suppress,retard, prevent, stop, or reverse a manifestation of symptoms. In otherembodiments, treatment of a neurodegenerative disorder is intended torelieve to some extent one or more of the symptoms associated with thedisorder. In these situations, the symptoms can be neuropsychiatricsymptoms, cognitive symptoms, and/or motor dysfunction. In otherembodiments, treatment of a neurodegenerative disorder is intended toreduce morbidity and mortality. In other embodiments, treatment of aneurodegenerative disorder is intended to improve quality of life.

In one embodiment, the disclosure relates to the use of anti-SEMA4Dbinding molecules, e.g., antibodies or antigen-binding fragments,variants, or derivatives thereof, as a medicament, in particular for usein the treatment of neurodegenerative disorders to improve the symptomsassociated with the disorder.

In accordance with the methods of the present disclosure, at least oneanti-SEMA4D binding molecule, e.g., an antibody or antigen bindingfragment, variant, or derivative thereof, or other biologic or smallmolecule as defined elsewhere herein can be used to promote a positivetherapeutic response with respect to the neurodegenerative disorder. A“positive therapeutic response” with respect to the neurodegenerativedisorder is intended to include an improvement in the symptomsassociated with the disorder. Such positive therapeutic responses arenot limited to the route of administration and can compriseadministration to the donor, the donor tissue (such as for example organperfusion), the host, any combination thereof, and the like. Inparticular, the methods provided herein are directed to inhibiting,preventing, reducing, alleviating, or lessening the progression of aneurodegenerative disorder in a patient. Thus, for example, animprovement in the disorder can be characterized as an absence ofclinically observable symptoms, a decrease in the incidence ofclinically observable symptoms, or a change in the clinically observablesymptoms.

Activities that change the symptoms associated with neurodegenerativedisorders can be detected and measured using in vivo mouse models. Incertain embodiments, a CVN mouse model can be employed. The CVN mouseincorporates mutations of AP precursor protein that are characteristicof familial Alzheimer's disease (AD) in three independent lineagestogether with a mutation that reproduces some of the conditions of braininflammation associated with AD (Colton et al., J Alzheimer's Dis.15:571-587, 2008; Van Nostrand et al., Stroke 41:S135-S138, 2010). TheCVN model displays some of the primary pathologies associated withAlzheimer's disease: AP plaques, hyperphosphorylated tau causingneurofibrillary tangles and cell death (neuronal loss), and consistentspatial memory impairment and neurovascular deficits. In comparison toother mouse mutants used for modeling Alzheimer's disease, the CVN Mouseshows more Alzheimer related pathologies at an earlier age. In otherembodiments, the YAC128 mouse model of Huntington's Disease (HD) can beemployed. YAC128 mice express the full-length mutant human huntingtingene (mHTT) and accurately recapitulate many of the signs and symptomsof HD. It should be appreciated that people skilled in the art willrecognize that other models have been described and usefully employedfor studies of disease mechanisms and treatment of symptoms inneurodegenerative disorders in the literature and that the presentdisclosure should not be limited to any one particular model.

The anti-SEMA4D binding molecules, e.g., antibodies or antigen bindingfragments, variants, or derivatives thereof or other biologics or smallmolecules can be used in combination with at least one or more othertreatments for neurodegenerative disorders; where the additional therapyis administered prior to, during, or subsequent to the anti-SEMA4Dbinding molecule, e.g., antibody or antigen binding fragment, variant,or derivative thereof, therapy. Thus, where the combined therapiescomprise administration of an anti-SEMA4D binding molecule, e.g., anantibody or antigen binding fragment, variant, or derivative thereof, incombination with administration of another therapeutic agent, themethods of the disclosure encompass coadministration, using separateformulations or a single pharmaceutical formulation, with simultaneousor consecutive administration in either order.

To apply the methods and systems of the disclosure in certainembodiments, samples or images from a patient can be obtained before or,after, or both before and after the administration of a therapycomprising an effective amount of an isolated binding molecule thatspecifically binds to Semaphorin-4D (SEMA4D), to a subject determined tohave a neurodegenerative disorder, or to a subject suspected of having aneurodegenerative disorder. In some cases, successive samples or imagescan be obtained from the patient after therapy has commenced, or aftertherapy has ceased, or both before and after therapy. Samples or imagescan, for example, be requested by a healthcare provider (e.g., a doctor)or healthcare benefits provider, obtained and/or processed by the sameor a different healthcare provider (e.g., a nurse, a hospital) or aclinical laboratory, and after processing, the results can be forwardedto yet another healthcare provider, healthcare benefits provider, or thepatient. Similarly, the measuring/determination of one or more scores,comparisons between scores, evaluation of the scores and treatmentdecisions can be performed by one or more healthcare providers,healthcare benefits providers, and/or clinical laboratories.

In certain aspects of any of the aforementioned procedures, theneurodegenerative disorder is selected from a group consisting ofAlzheimer's disease, Parkinson's disease, Huntington's disease, Downsyndrome, ataxia, amyotrophic lateral sclerosis (ALS), frontotemporaldementia (FTD), HIV-related cognitive impairment, CNS Lupus, mildcognitive impairment, or a combination thereof. In certain aspects ofany of the aforementioned procedures, the neurodegenerative disorder isAlzheimer's disease or Huntington's disease,

In some aspects, a healthcare provider can administer or instructanother healthcare provider to administer a therapy comprising aneffective amount of an isolated binding molecule that specifically bindsto Semaphorin-4D (SEMA4D), where the subject has, is determined to have,or is suspected to have, a neurodegenerative disorder. A healthcareprovider can implement or instruct another healthcare provider orpatient to perform the following actions: obtain a sample or image,process a sample or image, submit a sample or image, receive a sample orimage, transfer a sample or image, analyze or measure a sample or image,quantify a sample or image, provide the results obtained afteranalyzing/measuring/quantifying a sample or image, receive the resultsobtained after analyzing/measuring/quantifying a sample or image,compare/score the results obtained after analyzing/measuring/quantifyingone or more samples or images, provide the comparison/score from one ormore samples, obtain the comparison/score from one or more samples orimages, administer a therapy, e.g., an effective amount of an isolatedbinding molecule that specifically binds to Semaphorin-4D (SEMA4D),commence the administration of a therapy, cease the administration of atherapy, continue the administration of a therapy, temporarily interruptthe administration of a therapy, increase the amount of an administeredtherapeutic agent, decrease the amount of an administered therapeuticagent, continue the administration of an amount of a therapeutic agent,increase the frequency of administration of a therapeutic agent,decrease the frequency of administration of a therapeutic agent,maintain the same dosing frequency on a therapeutic agent, replace atherapy or therapeutic agent by at least another therapy or therapeuticagent, combine a therapy or therapeutic agent with at least anothertherapy or additional therapeutic agent.

In some aspects, a healthcare benefits provider can authorize or deny,for example, collection of a sample, processing of a sample, submissionof a sample, receipt of a sample, transfer of a sample, analysis ormeasurement a sample, quantification a sample, provision of resultsobtained after analyzing/measuring/quantifying a sample, transfer ofresults obtained after analyzing/measuring/quantifying a sample,comparison/scoring of results obtained afteranalyzing/measuring/quantifying one or more samples, transfer of thecomparison/score from one or more samples, administration of a therapyor therapeutic agent, commencement of the administration of a therapy ortherapeutic agent, cessation of the administration of a therapy ortherapeutic agent, continuation of the administration of a therapy ortherapeutic agent, temporary interruption of the administration of atherapy or therapeutic agent, increase of the amount of administeredtherapeutic agent, decrease of the amount of administered therapeuticagent, continuation of the administration of an amount of a therapeuticagent, increase in the frequency of administration of a therapeuticagent, decrease in the frequency of administration of a therapeuticagent, maintain the same dosing frequency on a therapeutic agent,replace a therapy or therapeutic agent by at least another therapy ortherapeutic agent, or combine a therapy or therapeutic agent with atleast another therapy or additional therapeutic agent.

In certain aspects of any of the aforementioned procedures, theneurodegenerative disorder is selected from a group consisting ofAlzheimer's disease, Parkinson's disease, Huntington's disease, Downsyndrome, ataxia, amyotrophic lateral sclerosis (ALS), frontotemporaldementia (FTD), HIV-related cognitive impairment, CNS Lupus, mildcognitive impairment, or a combination thereof. In certain aspects ofany of the aforementioned procedures, the neurodegenerative disorder isAlzheimer's disease or Huntington's disease.

In addition, a healthcare benefits provider can, e.g., authorize or denythe prescription of a therapy, authorize or deny coverage for therapy,authorize or deny reimbursement for the cost of therapy, determine ordeny eligibility for therapy, etc.

In some aspects, a clinical laboratory can, for example, collect orobtain a sample, process a sample, submit a sample, receive a sample,transfer a sample, analyze or measure a sample, quantify a sample,provide the results obtained after analyzing/measuring/quantifying asample, receive the results obtained afteranalyzing/measuring/quantifying a sample, compare/score the resultsobtained after analyzing/measuring/quantifying one or more samples,provide the comparison/score from one or more samples, obtain thecomparison/score from one or more samples, or other related activities.

In certain aspects of any of the aforementioned procedures, theneurodegenerative disorder is selected from a group consisting ofAlzheimer's disease, Parkinson's disease, Huntington's disease, Downsyndrome, ataxia, amyotrophic lateral sclerosis (ALS), frontotemporaldementia (FTD), HIV-related cognitive impairment, CNS Lupus, mildcognitive impairment, or a combination thereof. In certain aspects ofany of the aforementioned procedures, the neurodegenerative disorder isAlzheimer's disease or Huntington's disease.

In certain aspects, any of the aforementioned procedures can be used todetermine if a subject has a neurodegenerative disorder. In certainaspects, the neurodegenerative disorder is selected from a groupconsisting of Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Down syndrome, ataxia, amyotrophic lateral sclerosis (ALS),frontotemporal dementia (FTD), HIV-related cognitive impairment, CNSLupus, mild cognitive impairment, or a combination thereof. In certainaspects of any of the aforementioned procedures, the neurodegenerativedisorder is Alzheimer's disease or Huntington's disease,

In some aspects, a healthcare provider, clinical laboratory, or otherentity can, for example, collect or obtain an image, process an image,submit an image, receive an image, transfer an image, analyze or measurean image, quantify an image, provide the results obtained afteranalyzing/measuring/quantifying an image, receive the results obtainedafter analyzing/measuring/quantifying an image, compare/score theresults obtained after analyzing/measuring/quantifying one or moreimages, provide the comparison/score from one or more images, obtain thecomparison/score from one or more images, or other related activities.Images that can be used in such aspects include, but are not limited to,images obtained by angiography, ultrasound, computed tomography (CT),magnetic resonance imaging (MRI), positron emission tomography (PET),optical coherence tomography (OCT), near-infrared spectroscopy (NIRS),and NIR fluorescence. In certain embodiments, imaging techniques thathave been described in the literature can be used (Tardif et al. CircCardiovasc Imaging 4:319-333 (2011)).

VI. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering anti-SEMA4D binding molecules,e.g., antibodies, or antigen-binding fragments, variants, or derivativesthereof to a subject in need thereof are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe anti-SEMA4D binding molecule, e.g, antibody, or antigen-bindingfragment, variant, or derivative thereof, can be, for example, oral,parenteral, by inhalation or topical. The term parenteral as used hereinincludes, e.g., intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, rectal, or vaginal administration. Whileall these forms of administration are clearly contemplated as beingwithin the scope of the disclosure, an example of a form foradministration would be a solution for injection, in particular forintravenous or intraarterial injection or drip. A suitablepharmaceutical composition for injection can comprise a buffer (e.g.acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate),optionally a stabilizer agent (e.g. human albumin), etc. However, inother methods compatible with the teachings herein, anti-SEMA4D bindingmolecules, e.g., antibodies, or antigen-binding fragments, variants, orderivatives thereof can be delivered directly to the site of the adversecellular population thereby increasing the exposure of the diseasedtissue to the therapeutic agent.

As discussed herein, anti-SEMA4D binding molecules, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof can beadministered in a pharmaceutically effective amount for the in vivotreatment of neurodegenerative disorders. In this regard, it will beappreciated that the disclosed binding molecules can be formulated so asto facilitate administration and promote stability of the active agent.In certain embodiments, pharmaceutical compositions in accordance withthe present disclosure comprise a pharmaceutically acceptable,non-toxic, sterile carrier such as physiological saline, non-toxicbuffers, preservatives and the like. For the purposes of the instantapplication, a pharmaceutically effective amount of an anti-SEMA4Dbinding molecule, e.g., an antibody, or antigen-binding fragment,variant, or derivative thereof, shall be held to mean an amountsufficient to achieve effective binding to a target and to achieve abenefit, e.g., improve the symptoms associated with a neurodegenerativedisorder.

The pharmaceutical compositions used in this disclosure comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include, e.g., water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject disclosure, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1 M, e.g., about 0.05 Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives can also be present such as, for example,antimicrobials, antioxidants, chelating agents, and inert gases and thelike.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and can be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Suitable formulations foruse in the therapeutic methods disclosed herein are described inRemington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed.(1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, isotonic agents can be included, for example, sugars,polyalcohols, such as mannitol, sorbitol, or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an anti-SEMA4D antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,methods of preparation include vacuum drying and freeze-drying, whichyield a powder of an active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Thepreparations for injections are processed, filled into containers suchas ampoules, bags, bottles, syringes or vials, and sealed under asepticconditions according to methods known in the art. Further, thepreparations can be packaged and sold in the form of a kit. Sucharticles of manufacture can have labels or package inserts indicatingthat the associated compositions are useful for treating a subjectsuffering from, or predisposed to a disease or disorder.

Parenteral formulations can be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionscan be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in this disclosure can beorally administered in an acceptable dosage form including, e.g.,capsules, tablets, aqueous suspensions or solutions. Certainpharmaceutical compositions also can be administered by nasal aerosol orinhalation. Such compositions can be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, and/or other conventionalsolubilizing or dispersing agents.

The amount of an anti-SEMA4D binding molecule, e.g., antibody, orfragment, variant, or derivative thereof, to be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Thecomposition can be administered as a single dose, multiple doses or overan established period of time in an infusion. Dosage regimens also canbe adjusted to provide the optimum desired response (e.g., a therapeuticor prophylactic response).

In keeping with the scope of the present disclosure, anti-SEMA4Dantibodies, or antigen-binding fragments, variants, or derivativesthereof can be administered to a human or other animal in accordancewith the aforementioned methods of treatment in an amount sufficient toproduce a therapeutic effect. The anti-SEMA4D antibodies, orantigen-binding fragments, variants or derivatives thereof can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the disclosure with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.Those skilled in the art will further appreciate that a cocktailcomprising one or more species of anti-SEMA4D binding molecules, e.g.,antibodies, or antigen-binding fragments, variants, or derivativesthereof, of the disclosure can be used.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-SEMA4D binding molecule, e.g., antibody orantigen binding fragment, variant, or derivative thereof, that whenadministered brings about a positive therapeutic response with respectto treatment of a patient with a disease to be treated. In the case of aneurodegenerative disorder, a positive therapeutic response canalleviate symptoms of the disorder; decrease, reduce, retard or stop theincidence of symptoms; decrease, reduce, retard the severity ofsymptoms; inhibit, e.g., suppress, retard, prevent, stop, or reverse themanifestation of symptoms; relieve to some extent one or more of thesymptoms associated with the disorder; reduce morbidity and mortality;improve quality of life; or a combination of such effects.

Therapeutically effective doses of the compositions of the presentdisclosure, for the decrease in the incidence of symptoms, varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. In certain embodimentsthe patient is a human, but non-human mammals including transgenicmammals can also be treated. Treatment dosages can be titrated usingroutine methods known to those of skill in the art to optimize safetyand efficacy.

The amount of at least one anti-SEMA4D binding molecule, e.g., antibodyor binding fragment, variant, or derivative thereof, to be administeredis readily determined by one of ordinary skill in the art without undueexperimentation given the present disclosure. Factors influencing themode of administration and the respective amount of at least oneanti-SEMA4D binding molecule, e.g., antibody, antigen-binding fragment,variant or derivative thereof include, but are not limited to, theseverity of the disease, the history of the disease, and the age,height, weight, health, and physical condition of the individualundergoing therapy. Similarly, the amount of anti-SEMA4D bindingmolecule, e.g., antibody, or fragment, variant, or derivative thereof,to be administered will be dependent upon the mode of administration andwhether the subject will undergo a single dose or multiple doses of thisagent.

The disclosure also provides for the use of an anti-SEMA4D bindingmolecule, e.g., antibody of the disclosure, or antigen-binding fragment,variant, or derivative thereof, in the manufacture of a medicament fortreating a subject for treating a neurodegenerative disorder, whereinthe medicament is used in a subject that has been pretreated with atleast one other therapy. By “pretreated” or “pretreatment” is intendedthe subject has received one or more other therapies (e.g., been treatedwith at least one other neurodegenerative therapy) prior to receivingthe medicament comprising the anti-SEMA4D binding molecule, e.g.,antibody or antigen-binding fragment, variant, or derivative thereof“Pretreated” or “pretreatment” includes subjects that have been treatedwith at least one other therapy within 2 years, within 18 months, within1 year, within 6 months, within 2 months, within 6 weeks, within 1month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week,within 6 days, within 5 days, within 4 days, within 3 days, within 2days, or even within 1 day prior to initiation of treatment with themedicament comprising the anti-SEMA4D binding molecule, for example, themonoclonal antibodies VX15/2503, 67, or 76 disclosed herein, orantigen-binding fragment, variant, or derivative thereof. It is notnecessary that the subject was a responder to pretreatment with theprior therapy or therapies. Thus, the subject that receives themedicament comprising the anti-SEMA4D binding molecule, e.g., anantibody or antigen-binding fragment, variant, or derivative thereofcould have responded, or could have failed to respond, to pretreatmentwith the prior therapy, or to one or more of the prior therapies wherepretreatment comprised multiple therapies.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlang); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1: Testing the Effect of an Anti-SEMA4D BindingMolecule, e.g., an Antibody or Antigen-Binding Fragment, Variant, orDerivative Thereof, e.g., VX15/2503, 67, or 76 on Alzheimer's Disease(AD) in the CVN Mouse Model

Experimental Design.

The CVN model was used to study the effect of anti-SEMA4D antibody(e.g., MAb 67) on the pathologies and symptoms associated with AD. TheCVN mouse incorporates mutations of human Aβ precursor protein that arecharacteristic of familial Alzheimer's disease (AD) in three independentlineages together with a deletion of a gene (nitric oxide synthase-2) topromote neuroinflammatory mechanisms associated with AD (Colton et al.,J Alzheimer's Dis. 15:571-587, 2008; Van Nostrand et al., Stroke41:S135-S138, 2010).

The basic experimental design is shown in FIG. 1. Alzheimer's diseaseprone CVN mice (obtained from Charles River) were used to test theeffect of an anti-SEMA4D binding molecule on AD. At 10 weeks of age, themice were bled to obtain baseline serology levels. Between 10-12 weeksof age, the mice underwent behavioral pretesting to ensure they werecapable of participating in the study. Following randomization, the CVNmice were treated weekly with anti-SEMA4D (Mab-67) or isotype control(MAb 2B8) antibody (30 mg/kg, i.v.) from week 26 to 38 at which timethey were administered several behavioral tests. The behavioral testswere the open field test and the radial arm water maze.

Open Field Test—

Exploratory activity of the animal is studied in open field test at 10and 38 weeks of age for possible treatment induced hypo- orhyperactivity (control test) or other effect. Mice are brought to theexperimental room for at least 30 min acclimation to the experimentalroom conditions prior to testing. Activity chambers (Med Associates Inc,St Albans, Vt.; 27×27×20.3 cm) are equipped with IR beams. Mice areplaced in the center of the chamber and their behavior is recorded for30 min in 5-minute bins. Quantitative analysis is performed on thefollowing five dependent measures: total locomotion, locomotion in thecenter of the open field, rearing rate in the center, total rearingfrequency and velocity. Animals are tested at low-stress conditionswhere the light is lowered to approximately 10-30 lux of red light.

Radial Arm Water Maze—

At 11 and 39 weeks of age mice are brought to the experimental room forat least 30 min acclimation to the experimental room conditions prior totesting. Two-day radial-arm water maze has been described in detailpreviously (Alamed et al. 2006). Briefly, a six-arm maze is submerged ina pool of water, and a platform is placed at the end of one arm. Themouse receives 15 trials per day for 2 d and on each trial is started ina different arm while the arm containing the platform remains the samefor each mouse. The platform location of which remains constant over the2 d for each mouse at a given age, but this location changes for eachmouse between the 11 and 40 weeks of age testing time point. Usingvisual cues around the room, the mouse learns the position of the escapeplatform. The first 10 trials are considered training and alternatebetween a visible and a hidden platform. The final trials for day 1 andall trials on day 2 use a hidden platform. The number of errors(incorrect arm entries) was counted over a 1 min period. The errors areaveraged over three trials resulting in 10 blocks for the 2 d period.

Following the conclusion of behavioral testing, mice were sacrificed,and brain tissues were processed for formalin-fixed paraffin-embedded(FFPE) immunohistochemistry. In view of reports of a role for SEMA4D inthe induction of inhibitory GABAergic synapses (Kuzirian et al., JNeuroscience, 33:8961-8973• 8961, 2013) the density of vesicles andintensity of expression of Vesicular GABA Transporter (VGAT) wasdetermined in the Dentate Gyms of treated CVN mice. The Dentate Gyms isone of a few major centers of continued neurogenesis in the adult CNSand is thought to play a role in memory formation and retention. For alltests, statistical analysis was performed using the 2-way ANOVA test.

Anti-SEMA4D Reduces Anxiety-Like Behavior.

Exploratory activity was studied in groups of 12 AD prone CVN micetreated with anti-SEMA4D or isotype control. An open field test wasadministered at 38 weeks of age for possible treatment-induced effectson locomotor activity and anxiety-like behavior. Mice were placed in thecenter of a lighted chamber and their behavior was recorded for 30 minin six 5-minute time bins. Quantitative analysis was performed for totallocomotion (FIG. 2A) and locomotion in the center of the open field(FIG. 2B), which, as is known in the art, is a measure ofanxiety-related behavior.

The results showed that AD prone CVN mice treated weekly withanti-SEMA4D antibody manifest greater open field exploration and lessanxiety-like behavior (incursions into the center of the field) thanmice treated with control MAb 2B8. These results are shown in FIG. 2Aand FIG. 2B.

Anti-SEMA4D Improves Spatial Memory.

At 39 weeks of age, CVN mice treated with anti-SEMA4D or 2B8 isotypecontrol (n=9-13/group) were tested over 2-days in a radial-arm watermaze (Alamed et al. 2006, shown in FIG. 3A). Briefly, each mousereceived 15 trials per day (3 trials per block) on each of twoconsecutive days. Each trial was started from a different arm, while thearm containing the platform remained the same for each trial. Trialblocks on Day 1 alternated between a visible and hidden platform fortraining purposes. All trials on Day 2 were performed with a hiddenplatform to assess spatial memory. Day 1 was a training/learning periodand on day 2 the latency to find the platform was recorded.

The results showed that anti-SEMA4D antibody (MAb 67) administrationleads to a measurable decrease in latency suggesting improved spatialmemory as compared to the control (MAb 2B8-treated) cohort. The resultsare shown in FIG. 3B.

Anti-SEMA4D Decreases GABAergic Synapses.

FFPE brain tissue sections from Mab-67 or MAb-2B8 treated CVN and WTmice (n=9-13/group) were stained with anti-VGAT antibody to detectGABAergic synaptic vesicles. Percentages of VGAT-positive vesicle signaland VGAT signal intensities per vesicle were quantified within thedentate gyrus of all animals and normalized to total dentate gyrus areascanned.

The results showed that anti-SEMA4D antibody treatment of CVN AD miceleads to a trend of decreasing density of VGAT positive vesicles (FIG.4A) and a statistically significant decrease in VGAT staining intensitylevel per vesicle (FIG. 4B), a finding that suggests a role for SEMA4Din modulating GABAergic signaling in vivo and can provide mechanisticinsight into the behavioral effects observed in MAb 67 treated CVN mice.GABAergic signaling is associated with a heterogeneous class ofinhibitory neurons. As demonstrated in FIGS. 15A, 15B, 15C, and 15D ofExample 6 below, more significant effects of treatment with anti-SEMA4Dantibody on density of inhibitory neurons are observed when analysis isfocused on the subset of somatostatin-, NPY-, or NPY2R-positive neurons.

Example 2: Testing the Effect of an Anti-SEMA4D Binding Molecule, e.g.,an Antibody or Antigen-Binding Fragment, Variant, or Derivative Thereof,e.g., MAbs VX15/2503 or 67 on Huntington's Disease (HD) in the YAC128Mouse Model

Experimental Design.

A second experiment employing an in vivo YAC128 model was performed tostudy the effect of anti-SEMA4D antibody on the pathologies and symptomsassociated with HD. The basic experimental design was similar to thatshown in Example 1, and FIG. 1, above, but MAb (antibody) dosing in thiscase was performed weekly from week 6 to week 47 with between 13 and 22YAC128 or WT mice per group. The YAC128 mice were bred and maintained atUniversity of British Columbia, Centre for Molecular Medicine andTherapeutics.

Anti-SEMA4D Reduces Anxiety-Like Behavior in the YAC128 Mouse Model.

To assess anxiety during open-field exploration, MAb-treated mice wereplaced in the lower left corner of a 50×50 cm open grey acrylic box with20 cm tall sides in a room brightly lit by fluorescent ceiling lights.Open-field activity was recorded for 10 min by a ceiling-mounted videocamera. Entries into (FIG. 5A) and time spent in the center of the field(FIG. 5B) were scored as a measure of anxiety-like behavior.

In contrast to control treated animals where YAC128 mice manifestgreater anxiety-like behavior in open field exploration than wild type(WT) mice, there is no difference between WT and YAC128 mice treatedwith anti-SEMA4D antibody (MAb 67) indicating that MAb-67 amelioratesanxiety-like behavior in YAC128 mice. These results are shown in FIG. 5Aand FIG. 5B.

Anti-SEMA4D Improves Spatial Memory in the YAC128 Mouse Model.

To assess preference for a known object in a novel location, twodifferent novel objects were placed in the upper left and right handcorners of an open field box. Anti-SEMA4D-treated mice were introducedto the box in the lower left corner and recorded for 5 minutes (min) bya ceiling-mounted video camera, during which time the number ofinvestigations of the two novel objects were scored (Trial 1, FIG. 6A).Mice were then removed from the box for 5 min, and the object at the topright corner of the box was moved to the lower right corner of the box.Mice were reintroduced to the box and recorded for an additional 5 min(Trial 2, FIG. 6B). The percentage of the investigations, or nose pokes,to the target object (the one in the new location) relative to all nosepokes was computed.

In contrast to control or Mab 67-treated wild type (WT) animals thatpreferentially explore an object in a novel location in Trial 2,control-treated YAC128 mice do not recognize or show preference for theobject in a novel location. Treatment with anti-SEMA4D antibody restoresnormal novel object preference in YAC128 mice (p<0.01). This suggeststhat spatial memory in YAC128 mice was improved by Mab-67 treatment sothat they recognized that an object was in a novel location. The resultsare shown in FIG. 6A and FIG. 6B.

Anti-SEMA4D Prevents Cortical and Corpus Callosum Degeneration in YAC128Mice.

Free-floating brain tissue sections from 12 month-old MAb-treated YAC128and WT mice (n=13-21/group) were stained with anti-NeuN antibody.Cortical (FIG. 7A) and corpus callosum (FIG. 7B) volumes were determinedby tracing the perimeter of the defined structure in serial sectionsusing StereoInvestigator software (Microbrightfield) and volumes weredetermined using the Cavalieri principle.

The results show that treatment with anti-SEMA4D antibody inhibits thenormal disease related reduction in cortical and corpus callosum volumein YAC128 mice at 12 months of age. The results are shown in FIG. 7A andFIG. 7B.

Mab 67 Prevents Testicular Degeneration in YAC128 Mice.

Testicular degeneration is observed in male HD patients and isrecapitulated in male YAC128 mice. As shown in FIG. 8, treatment withanti-SEMA4D antibody prevents testicular degeneration in 12 month-oldYAC128 mice. It is possible that the effects of disease and anti-SEMA4Dantibody on both brain and testis reflect a common dependence onintracellular actin-dependent transport mechanisms in the normalfunction of these tissues.

Example 3: Examining Expression Patterns of SEMA4D, Plexin-B1, Plexin-B2and CD72 in the Rat CNS

To visualize the cell types within the CNS that express SEMA4D and itsreceptors plexin-B1, plexin-B2, and CD72, co-immunohistochemistry wasperformed on coronal spinal cord sections from naïve rats (FIG. 9A, FIG.9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H, FIG. 9I, FIG.9J, FIG. 9K, FIG. 9L, FIG. 9M, FIG. 9N, FIG. 9O, and FIG. 9P).Co-staining for the oligodendrocyte precursor cell marker, Nkx2.2, andSEMA4D (FIG. 9A-9C), plexin-B1 and the astrocytic marker, GFAP (FIG.9E-9G), plexin-B1 and CD72 (FIG. 9I-9K), and plexin-B1 and themicroglial marker, Iba1 (FIG. 9M-9O) was performed on spinal cordsections from naïve rats. In addition, all sections were stained withDAPI to visualize cellular nuclei (FIG. 9D, FIG. 9H, FIG. 9L, and FIG.9P). Slides were imaged at 60× magnification using an EXi-Aqua-14 bitcamera coupled to an Olympus Ix50 microscope.

The results in FIG. 9A-9P show that within the normal CNS, SEMA4D isrobustly expressed on Nkx2.2-positive oligodendrocyte precursors, whileits receptors, plexin-B1, plexin-B2 (data not shown), and CD72, areexpressed on multiple cell types and are especially prominent on GlialFibrillary Acidic Protein (GFAP)-positive astrocytes.

Example 4: Characterizing the Expression Patterns of Plexin-B1 andPlexin-B2 Receptors in the CVN Alzheimer's Disease Mouse Model

Homozygous bigenic CVN AD mice exhibit classical amyloid pathology andglial activation in the subiculum as compared to age-matched wild-typemice. Expression patterns of plexin-B1 were examined in the CVN mousemodel of Alzheimer's disease. CVN (also known as APPSwDI/NOS2−/−)bigenic mice harbor the amyloid precursor protein Swedish-Dutch-Iowamutant (APPSwDI) transgene and a targeted “null” mutation of the nitricoxide synthase 2 (Nos2, or inducible NOS, iNOS) locus. At 41 weeks ofage, CVN and wild-type control mice were sacrificed and processed forDAB immunohistochemistry. The results are shown in FIG. 10, with thewild-type mouse sections in the top panels and the CVN mouse sections inthe bottom panels). Sections were separately stained for amyloid-beta1-42 peptide (top and bottom panels at left), microglial marker Iba1(top and bottom center panels), or astrocyte marker GFAP (top and bottompanels at right). Slides were imaged at 20× magnification using a RetigaQICAM-12 bit camera coupled to an Olympus Ix50 microscope.

The results in FIG. 10 show that homozygous bigenic CVN mice(APPSwDI/NOS2−/−) develop classic amyloid pathology, microglialactivation, and astrogliosis (bottom panels).

Activated Astrocytes in CVN AD Mice Exhibit Enhanced Plexin-B1Expression as Compared to Age-Matched Wild-Type Mice.

At 41 weeks of age, CVN and wild-type control mice were sacrificed andprocessed for fluorescent co-immunohistochemistry (FIG. 11A). Sectionswere separately stained for the SEMA4D receptor plexin-B1 and astrocytemarker GFAP, and DAPI to visualize cellular nuclei. Composite images areshown in the left-most panels. Slides were imaged at 60× magnificationusing an EXi-Aqua-14 bit camera coupled to an Olympus Ix50 microscope.

As shown in FIG. 11A, co-immunohistochemical analyses of plexin-B1(second panels from left) and GFAP-positive astrocytes (third panelsfrom left) within the brains of CVN and age-matched wild-type micedemonstrates that astrocytic activation, as evidenced by increased GFAPmarker staining, positively correlates with enhanced co-registeredplexin-B1 expression, suggesting SEMA4D/plexin signaling participates inthe process of astrocyte activation.

Inhibiting SEMA4D Signaling in CVN AD Mice Restores Plexin-B2 Expressionas Compared to Age-Matched Wild-Type Mice.

To determine if blocking SEMA4D signaling in CVN AD mice would impactthe expression of plexin-B1 and/or its alternate cognate receptor,plexin-B2, in brain regions affected early in AD pathogenesis, 26week-old CVN and wild-type control mice were injected weekly with 30mg/kg anti-SEMA4D monoclonal antibody (67-2) or control IgG (2B8)intravenously for 13 weeks. At 41 weeks of age, mice were sacrificed andbrain tissue sections from MAb-treated mice were stained withanti-plexin-B1 or anti-plexin-B2 to detect whether anti-SEMA4D treatmentaltered cognate receptor expression in the setting of ongoing AD-relatedpathogenesis. Percentage of plexin-B1 and plexin-B2-positive signalswere quantified within the subiculum of all animals and normalized tototal subiculum area scanned.

As shown in FIG. 11B, anti-SEMA4D antibody treatment of CVN AD mice leadto a restoration in plexin-B2 (right graph) staining intensity levels tothose quantified in WT control mice, but no significant change inplexin-B1 levels (left graph) relative to age-matched CVN AD controlmice. These results suggest antibody-mediated SEMA4D inhibitionselectively leads to destabilization of plexin-B2 and/or impedes aSEMA4D-driven feed-forward mechanism that selectively promotes plexin-B2expression.

Example 5: Characterizing the Expression Patterns of Plexin-B2 Receptorin a YAC128 Huntington's Disease Mouse Model

YAC128 mice express the full-length mutant human huntingtin gene (mHTT)and accurately recapitulate many of the signs and symptoms of HD (seealso Example 2). Activated astrocytes in YAC128 Huntington disease miceexhibit enhanced plexin-B2 expression as compared to age-matchedwild-type mice. At 12 months of age, YAC128 mice and wild-type controlmice were sacrificed and processed for fluorescentco-immunohistochemistry (FIG. 12). Sections were co-stained forplexin-B2 (Plexin-B2, third panels from left), astrocyte marker GFAP(second panels from left) and DAPI to visualize cellular nuclei.Composite images are shown in the left-most panels. Slides were imagedat 60× magnification using an EXi-Aqua-14 bit camera coupled to anOlympus Ix50 microscope.

As shown in FIG. 12, co-immunohistochemical analyses of plexin-B2 andGFAP-positive astrocytes within the brains of YAC128 and wild-type micedemonstrates that astrocytic activation, as evidenced by increased GFAPmarker staining, again positively correlates with co-registered SEMA4Dreceptor expression. Plexin-B2, whose best characterized ligand isSEMA4C, also has an intermediate affinity for SEMA4D (Azzarelli R, etal. An antagonistic interaction between PlexinB2 and Rnd3 controls RhoAactivity and cortical neuron migration. Nature Commun. 2014; DOI:10.1038/ncomms4405)

Example 6: Examining the Mechanisms by which SEMA4D Signaling canModulate Astrocyte Function

The correlation between enhanced SEMA4D receptor expression andastrocytic activation in the setting of both AD and HD neurodegenerativedisease suggests that SEMA4D signaling plays a role in astrocytefunction and/or dysfunction. While not wishing to be bound by theory,this example provides evidence that SEMA4D signaling can participate inthe regulation of astrocytic function during responses to CNS injury,whether from acute or chronic stimuli. A schematic model supported bythe data is depicted in FIG. 13, which shows three mechanisms by whichSEMA4D signaling can potentially modulate astrocyte function. Thesethree mechanisms are discussed below.

Role of Astrocytes and OPC Support.

Plexin+ astrocytic processes interdigitate between SEMA4D+ NKX2.2+oligodendrocyte precursor cells (OPCs) and provide trophic support. InCNS disease, activated astrocytes upregulate Plexin expression andretract processes via SEMA4D signaling. Locally, this loss ofastrocyte:OPC proximity can result in diminished trophic support andincreased chemotaxis-driven OPC movement toward regions of damage, whilelack of astrocytic support at lesion site can impede OPC differentiationand remyelination.

To test this hypothesis, wild-type control rats were sacrificed andspinal cords were processed for fluorescent co-immunohistochemistry(FIG. 14). Sections were co-stained for SEMA4D (second panel from left),astrocyte marker GFAP (third panel from left) and DAPI to visualizecellular nuclei (right panel). Composite images are shown in theleft-most panels and the dotted box depicts a 1.67× magnified insetbelow. Slides were imaged at 60× magnification using an EXi-Aqua-14 bitcamera coupled to an Olympus Ix50 microscope.

As shown in FIG. 14, SEMA4D-expressing OPC are oriented in closeproximity with GFAP+ astrocyte processes. Given the role that astrocytesplay in facilitation of OPC survival and function, the juxtaposition ofSEMA4D-expressing OPCs and SEMA4D receptor-expressing astrocytessuggests that disease-related activation of astrocytes with associatedupregulation of plexin-B receptors and SEMA4D signaling can affect OPCfunction.

Role of Astrocytes in Neuronal Support.

In CNS disease, astrocytic activation leads to upregulation of Plexinexpression, increased SEMA4D signaling and process retraction, whichresults in a loss of neuronal axon guidance, decreased trophic support,and/or dysregulated glutamate uptake/release. Ultimately, depending uponseverity of disease stimulus, synapse loss and subsequent excitotoxicneuronal death can occur.

To determine if blocking SEMA4D signaling in CVN AD mice would impactsynaptic marker expression in brain regions affected early in ADpathogenesis, 26 week-old CVN and wild-type control mice were injectedweekly with 30 mg/kg anti-SEMA4D monoclonal antibody (67-2) or controlIgG (2B8) intravenously for 13 weeks. At 41 weeks of age, mice weresacrificed and brain tissue sections from MAb-treated mice were stainedwith anti-somatostatin antibody, anti-Neuropeptide-Y (NPY), anti-NPYreceptor 1 (NPY1R), or anti-NPY receptor 2 (NPY2R) to detect specificsubsets of inhibitory neurons that degenerate in early AD. Percentagesof somatostatin-positive signal were quantified within the subiculum,and NPY-, NPY1R-, or NPY2R-positive signal were quantified within thedentate gyrus for all animals and normalized, respectively, to totalsubiculum or dentate gyrus area scanned. The results are shown in FIG.15 A, FIG. 15B, FIG. 15C, and FIG. 15D.

FIGS. 15A-15D show that anti-SEMA4D antibody treatment of CVN AD miceleads to a restoration in somatostatin (FIG. 15A), NPY (FIG. 15B), andNPY2R (FIG. 15D) staining intensity levels to levels characteristic ofwild-type mice. Interestingly, agonists of NPY1R are reported to reducestress and anxiety, while antagonists specific for NPY2R reduce stressand anxiety (Markus Heilig. The NPY system in stress, anxiety anddepression. Neuropeptides 38 (2004) 213-224). As shown in FIG. 2A andFIG. 2B above, CVN mice treated with anti-SEMA4D exhibited reducedseverity in anxiety in open field tests, findings that correlated withnormalized (lower) NPY2R levels, while NPY1R levels were unchanged byanti-SEMA4D MAb treatment and remained higher than wild-type mice (FIG.15C). Hence, these changes in NPY receptor levels are concordant withreduced anxiety behaviors observed in anti-SEMA4D treated CVN mice, afinding that further supports a role for SEMA4D in modulation ofneurotransmission in vivo. It is noteworthy that downregulation of theinhibitory NPY neurotransmitter seen in the CVN AD model has also beenreported in cerebral cortex of patients with Alzheimer's disease (Beal,et al., Ann. Neurol. 20, 282-288 (1986).

Role of Astrocytes in Maintaining Blood-Brain Barrier Integrity.

As discussed elsewhere herein, astrocytic processes proximal to cerebralmicrovessels or pia are characterized by a high density of the waterchannel, aquaporin 4 (Aqp4). Astrocytic processes facing synapticregions are enriched in glutamate transporters, where the density ofAqp4 is comparatively low. CNS disease-induced astrocyte activationincreases SEMA4D signaling through Plexin, which leads to a retractionof astrocytic foot processes as evidenced by redistribution ofaquaporin-4. This results in dysregulation and permeability of the BBB,thereby facilitating endothelial inflammation and subsequent leukocyteentry into the CNS. In the setting of AD, Aqp4 staining intensitiessignificantly decrease in regions with significant amyloid plaqueburden.

To measure aquaporin-4 expression patterns in CVN AD mice as compared toage-matched wild-type mice, CVN and wild-type control mice weresacrificed at 41 weeks of age and processed for fluorescentco-immunohistochemistry. The results are shown in FIG. 16, withwild-type mice in the top panels and CVN mice in the lower panels.Sections were co-stained for aquaporin-4 (second panels from left),astrocyte marker GFAP (third panels from left) and DAPI (right panels)to visualize cellular nuclei. Composite images are shown in theleft-most panels. Slides were imaged at 60× magnification using anEXi-Aqua-14 bit camera coupled to an Olympus Ix50 microscope.

As shown in FIG. 16, Aqp4 staining in age-matched CVN mice revealed asignificant shift towards a diffuse pattern. This is in contrast toco-immunohistochemical analyses of Aqp4 and GFAP-positive astrocyteswithin the brains of wild-type mice, which demonstrate Aqp4 stainingpattern in the subiculum that is restricted to areas proximal tomicrovasculature. This alteration in Aqp4 distribution, or loss inpolarity, correlates with high astrocyte activation, as evidenced byincreased intensity in GFAP staining. Given the strong co-registrationin plexin-B1 staining in activated astrocytes (FIG. 11A and FIG. 11B)and the role of SEMA4D/plexin-B1 signaling in cellular processretraction, these data suggest that SEMA4D signaling can play a role inthe alteration of astrocyte polarity at the BBB interface duringdisease.

To analyze the impact of SEMA4D on BBB, a dynamic in vitro blood-brainbarrier (DIV-BBB) model was employed. Briefly, the model consists ofhollow polypropylene fibers that contain transcapillary 2 to 4-μmdiameter pores. The fibers were connected to a pulsatile pump thatfacilitates continuous flow of media, and experimental compounds throughthe fibers and normal stimulation of endothelial flow receptors. Humanbrain endothelial cells were inoculated into the luminal compartment andallowed to adhere to and coat the inside walls of the fibers, whilehuman astrocytes were seeded into the abluminal compartment bathing theoutside surface of the fibers. The endothelial cells and astrocytesinteract across the membrane to induce formation of a barrier with tightjunctions between endothelial cells. The integrity of this barrier canbe monitored continuously by measurement of trans-endothelial electricalresistance (TEER). Human brain endothelial cells were inoculated intothe luminal compartment and allowed to adhere to the polypropylenefibers, and human astrocytes were seeded separately on the abluminalsurface of the fibers. At peak TEER (approximately 14 days in vitro),0.5, 5, and 50 μg/ml recombinant SEMA4D was added successively at 12-hintervals. At 36 h after initial SEMA4D exposure, 250 μg/ml control IgG(MAb 2955; 1 DIV-BBB unit) or anti-SEMA4D (VX15/2503; 2 DIV-BBB units)was added and TEER measured for another 132 h. The results are shown inFIG. 17. Error bars represent standard deviation. The data shown arerepresentative of three independent experiments, with each demonstratingsimilar effects of SEMA4D and antibody on DIV-BBB integrity.

As shown in FIG. 17, the breakdown of BBB was reversed within 24 h bythe addition of anti-SEMA4D antibody (VX15/2503). Introduction of acontrol recombinant protein did not result in a decrease in TEER (datanot shown). Moreover, introduction of control IgG antibody (MAb 2955)did not affect SEMA4D-induced BBB compromise.

These data suggest that CNS disease-induced astrocyte activationincreases SEMA4D signaling through plexin-B1 and/or plexin-B2upregulation, which leads to a retraction of astrocytic foot processesas evidenced by redistribution of aquaporin-4. This results indysregulation and permeability of the BBB, thereby facilitatingendothelial inflammation and subsequent leukocyte entry into the CNS.

Role of SEMA4D Signaling in Promoting Astrocyte Activation.

Given the association of SEMA4D receptor expression and the astrocyteactivation marker GFAP, there exists the possibility that SEMA4Dsignaling can potentiate astrocyte activation, thereby providing a“feed-forward” mechanism during disease states. To examine the effectsof SEMA4D on astrocyte activation, primary cultures of rat astrocyteswere generated and treated with SEMA4D in isolation or in combinationwith thioacetamide (TAA) pretreatment, a well known hepatotoxic andhepatocarcinogenic agent that has been shown to induce plexin-B1expression in vivo (Lim, J. S., et al., (2006). Mol. Cell. Tox., 2(2),126-133). Rat primary astrocytes were pretreated for 4 h with TAAfollowed by soluble SEMA4D for 24 h. Cells were then fixed and stainedfor GFAP and scanned at 20× magnification. Error bars represent standarddeviation. “*”=p<0.05 by 1-way ANOVA with Bonferroni's MultipleComparison Test.

As shown in FIG. 18A, a significant enhancement in GFAP, an activationmarker for astrocytes, was observed upon addition of SEMA4D to cellspretreated with TAA, suggesting that SEMA4D signaling enhances astrocyteactivation.

In a second set of in vitro studies, primary rat astrocytes werecultured and treated with or without prostaglandin D2, a knownactivation factor produced by microglia in the CNS, followed by an 8 or24-h exposure to recombinant SEMA4D protein. Cells were fixed, processedfor phalloidin (F-actin) and DNAse (G-actin) histochemistry, andF-actin/G-actin area ratios were calculated for each treatmentcondition. Error bars represent standard deviation. “**”=p<0.01 by 2-wayANOVA with Bonferroni's Multiple Comparison Test.

As shown in FIG. 18B, PGD2-activated astrocytes exposed to recombinantSEMA4D undergo a globular to filamentous transition in their actincytoskeleton that is indicative of SEMA4D/Plexin-mediated signaling anda heightened astrocyte activation state.

Many modifications and other embodiments of the disclosures set forthherein will come to mind to one skilled in the art to which thesedisclosures pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the disclosures are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims and listof embodiments disclosed herein. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

What is claimed is:
 1. A method of treating a subject having a having,determined to have, or suspected of having a neuroinflammatory orneurodegenerative disorder, comprising administering to the subject aneffective amount of an isolated antibody or antigen-binding fragmentthereof that specifically binds to SEMA4D, wherein the antibody orantigen-binding fragment thereof comprises a variable heavy chain (VH)comprising VHCDRs 1-3 comprising SEQ ID NOs 6, 7, and 8, respectively,and a variable light chain (VL) comprising VLCDRs 1-3 comprising SEQ IDNOs 14, 15, and 16, respectively, and wherein the binding to SEMA4D actsto alleviate symptoms associated with the disorder.
 2. The method ofclaim 1, wherein the binding molecule modulates astrocyte-mediatedactivity of oligodendrocyte-myelin function.
 3. The method of claim 1,wherein the binding molecule modulates astrocyte-mediated synapticactivity, thereby preventing neural cell death.
 4. The method of claim1, wherein the binding molecule modulates astrocyte-mediated maintenanceof the integrity of the blood-brain barrier.
 5. The method of claim 1,wherein the VH and VL comprise, respectively, SEQ ID NO: 9 and SEQ IDNO: 17 or SEQ ID NO: 10 and SEQ ID NO:
 18. 6. The method of claim 1,wherein the neurodegenerative disorder is selected from a groupconsisting of Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Down syndrome, ataxia, amyotrophic lateral sclerosis (ALS),frontotemporal dementia (FTD), HIV-related cognitive impairment, CNSLupus, mild cognitive impairment, or a combination thereof.
 7. Themethod of claim 1, wherein the antibody or fragment thereof isadministered to the subject in combination with at least one or moreother treatments for neuroinflammatory or neurodegenerative disorders,and wherein the other treatment is administered prior to, during, orsubsequent to administering the antibody or fragment thereof to thesubject.
 8. The method of claim 1, wherein the subject is human.
 9. Amethod of protecting inhibitory neurons from degeneration in earlyAlzheimer's disease, comprising administering to a subject having,determined to have, or suspected of having early Alzheimer's disease aneffective amount of an isolated antibody or antigen-binding fragmentthereof that specifically binds to SEMA4D, wherein the antibody orantigen-binding fragment thereof comprises a variable heavy chain (VH)comprising VHCDRs 1-3 comprising SEQ ID NOs 6, 7, and 8, respectively,and a variable light chain (VL) comprising VLCDRs 1-3 comprising SEQ IDNOs 14, 15, and 16, respectively, and wherein the antibody orantigen-binding fragment thereof restores the number of somatostatinpositive neurons, NYP-positive neurons, or both in the subject.
 10. Themethod of claim 9, wherein the VH and VL comprise, respectively, SEQ IDNO: 9 and SEQ ID NO: 17 or SEQ ID NO: 10 and SEQ ID NO:
 18. 11. Themethod of claim 9, wherein the antibody or fragment thereof modulatesastrocyte-mediated synaptic activity.
 12. The method of claim 9, whereinthe antibody or fragment thereof modulates astrocyte-mediatedmaintenance of the integrity of the blood-brain barrier.
 13. The methodof claim 9, wherein administration of the antibody or fragment thereofto the subject reduces the rate of neuronal cell death in the subject.14. The method of claim 9, wherein the antibody or fragment thereof isadministered to the subject in combination with at least one or moreother treatments for neuroinflammatory or neurodegenerative disorders,and wherein the other treatment is administered prior to, during, orsubsequent to administering the antibody or fragment thereof to thesubject.
 15. The method of claim 9, wherein the subject is human.